Agency: Cordis | Branch: FP7 | Program: CP-CSA-Infra | Phase: INFRA-2012-1.1.4. | Award Amount: 12.42M | Year: 2013
The mouse shows great similarities in development, physiology and biochemistry to humans, which makes it a key model for research into human disease. The major challenges for mouse functional genomics in the 21st century are to: Develop a series of mutant alleles for every gene in the mouse genome Determine the phenotypic consequences of each mutation Identify mouse models for the complete disease spectrum in humans To further develop and exploit the emerging mouse mutant resource, mouse models must be preserved and made available to the European biomedical research community. To this effect, the Infrafrontier-I3 project brings together the leading European centers for systemic phenotyping of mouse mutants and the European Mouse Mutant Archive network. The Infafrontier-I3 partners aim to meet the future challenges presented by phenotyping, archiving and disseminating mouse models in the ERA as follows: Contribute to resource development by archiving of 1215 new mouse mutant lines Provide free of charge Transnational Access to mouse production and 1st line phenotyping capacities Offer a specialized axenic service to produce, maintain and to distribute germ-free mice Provide user friendly accession of Infrafrontier services, extensive manual data curation and cross referencing with other mouse database Improve user services by developing novel phenotyping and cryopreservation SOPs and by refining innovative research instrumentation Engage with the user community using a wide range of PR activities, a dedicated user meeting and an industry liaison workshop Offer state of the art cryopreservation and phenotyping training courses Benchmark Infrafrontier services with other major repositories The comprehensive physical and data resources that will be generated by Infrafrontier-I3 will contribute to link basic biomedical research to medical applications and thereby drive innovation and support the Europe 2020 Strategy.
Based on power analyses that assumed a normal distribution, a 20% change in mean and 15% variation, we determined that at least 9 mice per group would be needed for behavioral experiments. This was adhered to as far as possible, except in cases where mice had to be removed owing to misplaced injections or lost headcaps. Mice were randomly assigned to groups and attempts were made to balance groups according to variables such as age and housing condition. The investigators were not blinded to allocation during experiments, but were blinded to outcome assessment for all behavioral experiments. Mice were used in all experiments. For experiments involving Cre lines, mice were crossed for several generations to C57 mice before using. All wild-type mice were C57BL/6 mice obtained from The Jackson Laboratory (Bar Harbour, ME). For all behavioural experiments except those involving Htr2cCre mice, male mice ranging in age from 8–16 weeks were used. Female Htr2cCre mice were used in chemogenetic manipulations. Both male and female mice aged 6–20 weeks were used for slice electrophysiology and anatomical tracing experiments. All behavioural studies or tissue collection for ex vivo slice electrophysiology were performed during the light cycle. All behavioural experiments in Htr2ccre mice were conducted at the University of Aberdeen and in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. All in vivo electrophysiology experiments were conducted in accordance with all rules and regulations at the National Institute for Alcohol Abuse and Alcoholism at the National Institutes of Health. All other procedures were conducted in accordance with the National Institutes of Health guidelines for animal research and with the approval of the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill. All animals were group housed on a 12 h light cycle (lights on at 7 a.m.) with ad libitum access to rodent chow and water, unless described otherwise. CRF-ires-Cre (Crfcre) were provided by Bradford Lowell (Harvard University) and were previously described21. C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbour, ME). To visualize CRF-expressing neurons, CrfCre mice were crossed with either an Ai9 or a Cre-inducible L10-GFP reporter line (Jackson Laboratory)22 to produce CRF-Ai9 or CRF-L10GFP progeny, referred to throughout the manuscript as CRF-reporters. SertCre mice (from GENSAT) were a generous gift from Bryan Roth. Htr2cCre mice were supplied by Lora Heisler and are described in detail elsewhere7. Male mice were used for in vivo optogenetic behavioural experiments and for assessing the involvement of BNST CRF neurons on fluoxetine-induced enhancement of fear. Female 5-HT -Cre mice were used in chemogenetic manipulations. Both male and female mice were used for slice electrophysiology and anatomical tracing experiments. All behavioural studies or tissue collection for ex vivo slice electrophysiology were performed during the light cycle. All AAV viruses except INTRSECT constructs were produced by the Gene Therapy Center Vector Core at the University of North Carolina at Chapel Hill and had titres of >1012 genome copies per ml. For ex vivo and in vivo optical experiments, mice were injected with rAAV5-ef1α-DIO-hChR2(H134R)-eYFP or rAAV5-ef1α-DIO-eYFP as a control. Red IX retrobeads (Lumafluor) were used to fluorescently label LH- and VTA-projecting BNST neurons during ex vivo slice electrophysiology recordings. The retrograde tracer Fluoro-Gold (Fluorochrome) was used for anatomical mapping. Choleratoxin B (CTB) 555 and CTB 657 retrograde tracers (Invitrogen; C34776, and C34778, respectively) diluted to 0.5% (w/v) in sterile PBS were used per injection site for anatomical mapping of collateral projections from BNST to LH and VTA. For chemogenetic manipulations, mice were injected with 400 nl of rAAV8-hsyn-DIO-hM3D(Gq)-mCherry, rAAV8-hsyn-DIO-hM4D(Gi)-mCherry, or rAAV8-hsyn-DIO-mCherry bilaterally. HSV-hEF1α-mCherry, HSV-ef1α-LSL1-mCherry-IRES-flpo, and HSV-ef1α-IRES-Cre (supplied by Rachel Neve at the McGovern Institute for Brain Research at MIT) were injected bilaterally into the VTA and LH at a volume of 500 nl per site. The INTRSECT construct AAVdj-hSyn-Con/Foff-hChR2(H134R)-EYFP was infused at 500 nl per side into the BNST. All AAV constructs had viral titres >1012 genome particles per ml. All surgeries were conducted using aseptic technique. Adult mice (2–5 months) were deeply anaesthetized with 5% isoflurane (v/v) in oxygen and placed into a stereotactic frame (Kopf Instruments) while on a heated pad. Sedation was maintained at 1.5–2.5% isoflurane during surgery. An incision was made down the midline of the scalp and a craniotomy was performed above the target regions and viruses and fluorescent tracers were microinjected using a Neuros Hamilton syringe at a rate of 100 nl min−1. After infusion, the needle was left in place for 10 min to allow for diffusion of the virus before the needle was slowly withdrawn. Injection coordinates (in mm, midline, Bregma, dorsal surface): BNST (±1.00, 0.30, −4.35), LH (±0.9 to 1.10, −1.7, −5.00 to −5.2), VTA (−0.3, −2.9, −4.6), DR (0.0, −4.65, −3.2 with a 23° angle of approach). When using retrobeads, injection volumes into the LH and VTA were 300 nl and 400 nl, respectively. Fluoro-Gold injection volumes were 200 nl per target site. CTB volumes were 200 nl per target site. An optical fibre was implanted in the BNST (±1.00, 0.20, −4.15) at a 10° angle for in vivo photostimulation studies. After fibre implantation, dental cement was used to adhere the ferrule to the skull. Following surgery, all mice returned to group housing. Mice were allowed to recover for at least 3 weeks before being used for chemogenetic behavioural studies, or 6 weeks for in vivo optogenetic studies. RS-102221, 5-HT and mCPP were from Tocris (Bristol, UK). For electrophysiology experiments, RS-102221 was made up to 100 mM in DMSO and then diluted to a final concentration of 5 μM in aCSF. 5-HT and mCPP were stocked at 10 and 20 mM, respectively, in ddH O and diluted to their final concentations in aCSF. For electrophysiology experiments, clozapine-N-oxide (CNO; from Bryan Roth) was stocked at 100 mM in DMSO and diluted to 10 μM in aCSF. For behaviour experiments, CNO was dissolved in 0.5% DMSO (in 0.9% saline) to a concentration of 0.1 mg ml−1 or 0.3 mg ml−1 and injected at 10 ml per kg for a final concentration of 1 or 3 mg per kg, i.p. Fluoxetine (Sigma) was made up in 0.9% NaCl to a concentration of 1 mg ml−1 and then injected at 10 ml per kg for a final concentration of 10 mg per kg, i.p. Surgical procedures. Mice were anaesthetized with 2% isoflurane (Baxter Healthcare, Deerfield, IL) and implanted with 2 × 8 electrode (35 μm tungsten) micro-arrays (Innovative Neurophysiology, Durham, NC) targeted at the BNST (ML: 0.8 mm, AP: ± 0.5 mm, and DV: −4.15 mm relative to Bregma). Following surgery, mice were singly housed and allowed at least one week to recover before behavioural testing. Fear conditioning. Fear conditioning took place in 27 × 27 × 11 cm conditioning chambers (Med Associates, St. Albans, VT), with a metal-rod floor (context A) and scented with 1% vanilla. Mice received 5 parings of a pure tone CS with a 0.6 mA foot shock. 24 h following conditioning, mice underwent a CS recall test (10 presentations of the CS alone, 5 s ITI), which was conducted in a Plexiglas cylinder (20 cm diameter) and scented with 1% acetic acid (context B). Stimulus presentations for both tests were controlled by MedPC (Med Associates, St. Albans, VT). Cameras were mounted overhead for recording freezing behaviour, which was scored automatically using CinePlex Behavioural Research System software (Plexon, Dallas, TX). Electrophysiological recording and single unit analysis. Electrophysiological recording took place during both fear conditioning and CS recall tests. Individual units were identified and recorded using Omniplex Neural Data Acquisition System (Plexon, Dallas, TX). Neural data was sorted using Offline Sorter (Plexon, Dallas, TX). Waveforms were isolated manually, using principal component analysis. To be included in the analyses, spikes had to exhibit a refractory period of at least 1 ms. Autocorrelograms from simultaneously recorded units were examined to ensure that no cell was counted twice. Single units were analysed by generating perievent histograms (3 s bins) of firing rates from 30 s before CS onset until 30 s after CS offset (NeuroExplorer 5.0, Nex Technologies, Madison, AL). Firing rates were normalized to baseline (30 s before CS onset) using z-score transformation. Analysis included a total of 139 cells over three days of recording. Data reported for raw firing rates include only putative principal neurons (<10 Hz). The formula for computing the suppression ratio was (average freezing rate) / (average freezing rate + average movement rate). Each cell was calculated individually. A value of 0.5 = no change in rate). Ex vivo slice electrophysiology. Brains were sectioned at 0.07 (mm per s) on a Leica 1200S vibratome to obtain 300 μm coronal slices of the BNST, which were incubated in a heated holding chamber containing normal, oxygenated aCSF (in mM:124 NaCl, 4.4 KCl, 2 CaCl , 1.2 MgSO , 1 NaH PO , 10.0 glucose, and 26.0 NaHCO ) maintained at 30 ± 1 °C for at least 1 h before recording. Slices were transferred to a recording chamber (Warner Instruments) submerged in normal, oxygenated aCSF maintained at 28–30 °C at a flow rate of 2 ml min−1. Neurons of the BNST were visualized using infrared differential interference contrast (DIC) video-enhanced microscopy (Olympus). Borosilicate electrodes were pulled with a Flaming-Brown micropipette puller (Sutter Instruments) and had a pipette resistance between 3–6 MΩ. Signals were acquired via a Multiclamp 700B amplifier, digitized at 10 kHz and analysed with Clampfit 10.3 software (Molecular Devices, Sunnyvale, CA, USA). In SertCre or CrfCre mice, fluorescently labelled neurons expressing ChR2 were visualized and stimulated with a blue (470 nm) LED using a 1 Hz, 2 Hz, 5 Hz, 10 Hz, and 20 Hz stimulation protocol with a pulse width of 0.5 ms. Evoked action potentials were recorded in current clamp mode using a potassium gluconate based internal solution (in mM: 135 K+ gluconate, 5 NaCl, 2 MgCl , 10 HEPES, 0.6 EGTA, 4 Na ATP, 0.4 Na GTP, pH 7.3, 285–290 mOsmol). In CrfCre mice with ChR2 in the BNST and retrograde tracer beads in the VTA or LH, we visualized non-ChR2-expressing, beaded neurons using green (532 nm) LED. Recordings were conducted in voltage clamp mode using a caesium-methansulfonate (Cs-Meth) based internal solution (in mM: 135 caesium methanesulfonate, 10 KCl, 1 MgCl , 0.2 EGTA, 2 QX-314, 4 MgATP, 0.3 GTP, 20 phosphocreatine, pH 7.3, 285–290 mOsmol) so that we could detect EPSCs (−55 mV) and IPSCs (+10 mV) in the same neuron. After confirming the absence of a light-evoked EPSC signal, we measured light-evoked IPSCs during a single 5-ms light pulse of 470 nm. In a subset of these experiments, SR95531 (GABAzine, 10 μM) was bath applied for 10 min to block IPSCs. Crf-reporter mice were injected with retrograde tracer beads into the VTA (ML −0.5, AP −2.9, DV −4.6). We then recorded from beaded (VTA-projecting) and non-beaded (non-projecting) CRF neurons in the BNST. Acute drug effects were determined in current clamp mode in the presence of TTX using a potassium gluconate-based internal solution. After a 5-min stable baseline was established, 5-HT (10 μM) or mCPP (20 μM) was bath applied for 10 min while recording changes in membrane potential. The difference in membrane potential between baseline and drug application at peak effect (delta or Δ MP) was later determined. In a subset of mCPP experiments, slices were incubated with RS-102221 (5 μM) for at least 20 min before experiments began. Spontaneous inhibitory postsynaptic currents (sIPSCs) were assessed in voltage clamp using a potassium-chloride gluconate-based intracellular solution (in mM: 70 KCl, 65 K+-gluconate, 5 NaCl, 10 HEPES, 0.5 EGTA, 4 ATP, 0.4 GTP, pH 7.2, 285–290 mOsmol). IPSCs were pharmacologically isolated by adding kynurenic acid (3 mM) to the aCSF to block AMPA and NMDA receptor-dependent postsynaptic currents. The amplitude and frequency of sIPSCs were determined from 2 min recording episodes at −70 mV. The baseline was averaged from the 4 min preceding the application of 5-HT (10 μM) or mCPP (10 μM) for 10 min. In a subset of these experiments, RS-102221 (5 μM) was added to the aCSF and slices were incubated in this drug solution for at least 20 min before experiments began. For miniature IPSCs (mIPSCs), TTX was included in the aCSF to block network activity. In SertCre::ChR2BNST mice with retrograde tracer beads in the VTA, sIPSCs were recorded as described above. After achieving a stable baseline, a 10 s, 20 Hz photostimulation was applied. For assessment of spontaneous excitatory postsynaptic currents (sEPSCs), a caesium gluconate-based intracellular solution was used (in mM: 135 Cs+-gluconate, 5 NaCl, 10 HEPES, 0.6 EGTA, 4 ATP, 0.4 GTP, pH 7.2, 290–295 mOsmol). AMPA -mediated EPSCs were pharmacologically isolated by adding 25 μM picrotoxin to the aCSF. sEPSC recordings were acquired in 2 min recording blocks at −70 mV. Electrodes were fabricated as previously described and cut to 50–100 μm in length23. Animal and slice preparation were as described above for electrophysiology and slices were perfused on the rig in ACSF. Using a custom-built potentiostat (University of Washington Seattle), 5-HT recordings were made in the BNST using TarHeel CV written in laboratory view (National Instruments). Briefly a triangular waveform (−0.1 V to 1.3 V with a 10% phase shift at 1,000 V per s, versus Ag/AgCl) was applied to the carbon fibre electrode at a rate of 10 Hz. Slices were optically stimulated with 20 5-ms blue (490 nm) light pulses at a rate of 20 Hz down the submerged 40× objective. 10 cyclic voltammograms were averaged before optical stimulation for background subtraction. Voltammograms were digitally smoothed one time with a fast Fourier transform following data collection and analysed with HDCV (UNC Chapel Hill). Fluoxetine (10 μM) was bath applied following a stable baseline (20 min). For chemogenetic manipulations, mice were transported to a holding cabinet adjacent to the behavioural testing room to habituate for at least 30 min before being pretreated with CNO (3 mg per kg, i.p. for CrfCre mice and 1 mg per kg, i.p. for Htr Cre mice). All behavioural testing began 45 min following CNO treatment, with the exception of fear conditioning training, which occurred 30 min after a CNO injection. When assessing the effect of fluoxetine on fear conditioning, fluoxetine (10 mg per kg, i.p.), or vehicle, was administered 1 h before training (30 min before CNO treatment). For optogenetic manipulations, mice received bilateral stimulation (473 nm, ~10 mW, 5 ms pulses, 20 Hz) when specified. Unless specified, all equipment was cleaned with a damp cloth between mouse trials. All sessions were video recorded and analysed using EthoVision software (Noldus Information Technologies) except where noted. Mice were placed in the centre of an elevated plus maze and allowed to explore during a 5 min session. Light levels in the open arms were ~14 lux. During optogenetic manipulations mice received bilateral stimulation during the entire 5 min session. Mice that left the maze were excluded from analysis (n = 2 control, 1 ChR2 from optogenetic experiments). Mice were placed into the corner of a white Plexiglas open field arena (25 × 25 × 25 cm) and allowed to freely explore for 30 min. The centre of the open field was defined as the central 25% of the arena. For optogenetic studies the 30 min session was divided into three 10-min epochs consisting of stimulation off, stimulation on, and stimulation off periods. 48 h before testing, mice were provided with access to a single piece of Froot Loops cereal (Kellogg’s) in their home cage. 24 h before testing, home cage chow was removed and mouse body weights were recorded. Water remained available ad libitum. Beginning at least one hour before testing, mice transferred to new clean cages so they were singly housed for the test session and body weights were recorded. During the test session mice were placed into an arena (25 × 25 × 25 cm) that contained a single Froot Loop on top of a piece of circular filter paper. Mice were monitored by a live observer and the latency for the mouse to begin eating the pellet was measured, allowing up to 10 min. All mice began eating within this time. Following the initiation of feeding, mice were removed from the arena and placed back into their home cages. Mice were then provided with 10 min of access to a pre-weighed amount of Froot Loops for a post-test feeding session. After this 10 min post-test, the remaining Froot Loops were weighed and mice were returned to ad libitum home cage chow. Mice were returned to group housing at the end of this session. For optogenetic experiments, mice received constant 20 Hz optical stimulation during both the latency to feed assay and the 10 min post-test. During optogenetic experiments, one control mouse did not feed during the 10 min NSF session and was excluded from the results. SertCre mice were food deprived for 24 h. On the day of the experiment, mice were acclimated to the behaviour room for 1 h. A single pre-weighed food pellet was placed in the home cage and the mice were allowed to eat for 10 min during optogenetic stimulation. At the end of the experimental session, the pellet was removed and weighed and mice were given ad libitum access to food. Htr Cre mice were acclimated in metabolic chambers (TSE Systems, Germany) for 2 days before the start of the recordings. After acclimation, mice were food deprived for 24 h. Following fasting, mice received an i.p. injection of CNO 30 min before food presented again. Mice were recorded for 12 h with the following measurements being taken every 30 min: water intake, food intake, ambulatory activity (in x and z axes), and gas exchange (O and CO ) (using the TSE LabMaster system, Germany). Energy expenditure was calculated according to the manufacturer’s guidelines (PhenoMaster Software, TSE Systems). We used a three-day protocol to assess both cued and contextual fear recall. On the first day, mice were placed into a fear conditioning chamber (Med Associates) that contained a grid floor and was cleaned with a scented paper towel (19.5% ethanol, 79.5% H O, 1% vanilla). After a 3 min baseline period, mice were exposed to a 30 s tone (3 kHz, 80 dB) that co-terminated with a 2 s scrambled foot shock (0.6 mA). A total of 5 tone-shock pairings were delivered with a random inter-tone interval (ITI) of 60–120 s. For optogenetic studies, light stimulation occurred only during the 30-s tones of this session. Following delivery of the last foot shock, mice remained in the conditioning chamber for a 2-min consolidation period. 24 h later, mice were placed into a separate conditioning box (Med Associates) that contained a white Plexiglas floor, a striped pattern on the walls, and was cleaned and scented with a 70% ethanol solution. After a 3 min baseline period, mice were presented with 10 tones (30 s, 3 kHz, 80 dB) with a 5 s ITI. Mice remained in the chamber after the last tone for a two-minute consolidation period. 24 h later (48 h after training), mice were returned to the original training chamber for 5 min. For each session, freezing behaviour was hand-scored every 5 s by a trained observer blinded to experimental treatment as described previously24. Freezing was defined as a lack of movement except as required for respiration. All mice used for behavioural and anatomical tracing experiments were anesthetized with Avertin and transcardially perfused with 30 ml of ice-cold 0.01 M PBS followed by 30 ml of ice-cold 4% paraformaldehyde (PFA) in PBS. Brains were extracted and stored in 4% PFA for 24 h at 4 °C before being rinsed twice with PBS and stored in 30% sucrose and PBS until the brains sank. 45 μm slices were obtained on a Leica VT100S and stored in 50/50 PBS/Glycerol at −20 °C. DREADD or ChR2-containing sections were mounted on slides, allowed to dry, coverslipped with VectaShield (Vector Labs, Burlingame, CA), and stored in the dark at 4 °C. We stained free-floating dorsal raphe sections using indirect immunofluorescence sequentially for first tryptophan hydroxylase (TPH) and Fluoro-Gold (FG) and then c-fos. For TPH/FG, we washed sections 3× for 5 min with 0.01 M PBS, permeabilized them for 30 min in 0.5% Triton/0.01 M PBS, and washed the sections again 2× with 0.01 M PBS. We blocked the sections for 1 h in 0.1% Triton/0.01 M PBS containing 10% (v/v) normal donkey serum and 1% (w/v) bovine serum albumin (BSA). We then added primary antibodies (1:500 mouse anti-TPH (Sigma Aldrich T0678) and 1:3,000 guinea-pig anti-Fluoro-Gold (Protos Biotech NM101)) to blocking buffer and incubated the sections overnight at 4 °C. The next day, we washed the sections 3× for 5 min with 0.01 M PBS, then incubated them with 1:500 with Alexa Fluor 647-conjugated donkey anti-mouse and Alexa Fluor 488-conjugated donkey anti-guinea pig secondary antibodies for 2 h at room temperature, and washed the sections 4× for 5 min with 0.01 M PBS. We then proceeded directly to the c-fos tyramide signal amplification based immunofluorescent staining. We permeabilized the sections in 50% methanol for 30 min, then quenched endogenous peroxidase activity in 3% hydrogen peroxide for 5 min. Followed by two 10 min washes in 0.01 M PBS, we blocked the sections in PBS containing 0.3% Triton X-100 and 1.0% BSA for 1 h. c-fos primary antibody (Santa Cruz Biotechnology, -sc-52) was added to sections at 1:3,000 and sections were incubated for 48 h at 4 °C. On day 3, we washed the sections in TNT buffer (0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, 0.05% Tween-20) for 10 min, blocked in TNB buffer (0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, 0.5% blocking reagent – PerkinElmer FP1020) buffer for 30 min. We then incubated the sections in secondary antibody (goat anti-rabbit HRP-conjugated PerkinElmer) 1:200 in TNB buffer for 30 min, washed the sections in TNT buffer 4× for 5 min, and then incubated the sections in Cy3 dye diluted in TSA amplification diluents for 10 min. We washed the sections 2× in TNT buffer, mounted them on microscope slides. We coverslipped the slides using Vectashield mounting medium. We acquired 4–5 of 2 × 4 tiled z-stack (5 optical slices comprising 7 μm total) images of the dorsal raphe from each naive and shock mouse on a Zeiss 800 upright confocal microscope. Scanning parameters and laser power were matched between groups. Images were preprocessed using stitching and maximum intensity projection and then analysed using an advanced processing module in Zeiss Zen Blue that allows nested analysis of multiple segmented fluorescent channels within parent classes. Double-labelled and triple-labelled cells were validated in a semi-automated fashion. At least 4 sections per mouse were counted in this way. One mouse was identified as a significant outlier in the shock group and was excluded from further analysis. To verify expression of ChR2-expressing fibres in the BNST originating from DRN serotonergic neurons, 300 μm slices used for ex vivo electrophysiological recordings containing the DRN and BNST were stored in 4% paraformaldehyde at 4 °C for 24 h before being rinsed with PBS, mounted, and coverslipped with Vectashield mounting medium. Images showing eYFP fluorescence from the DRN and BNST were obtained on a Zeiss 800 upright confocal microscope using a 10× objective and tiled z stacks. To validate the INTRSECT construct, mice received injections of HSV-hEF1α-mCherry or HSV-ef1α-LSL1-mCherry-IRES-flpo to both the LH and VTA bilaterally (n = 4 and 5, respectively). Both groups received AAVDJ-hSyn-Cre-on/Flp-off-hChR2(H134R)-EYFP to the BNST bilaterally. Six weeks following injection, mice were perfused and tissue was collected as described above. To visualize YFP expression in the BNST of CrfCre::IntrsectBNST mice, free-floating slices containing the BNST were rinsed three times with PBS for 5 min each. Slices were then incubated in 50% methanol for 30 min then incubated in 3% hydrogen peroxide for 5 min. Following three 10-min washes in PBS, slices were incubated in 0.5% Triton X-100 for 30 min followed by a 10 min PBS wash. Slices were blocked in 10% normal donkey serum/0.1% Triton X-100 for 1 h, and then they were incubated overnight at 4°C with a primary chicken anti-GFP antibody (GFP-1020, Aves) at 1:500 in blocking solution. Following primary incubation, slices were rinsed three times with 0.01M PBS for 10 min each and incubated with a fluorescent secondary antibody (AlexaFluor 488 donkey anti-chicken) at 1:200 in PBS for 2 h at room temperature. Slices were then rinsed with four 10-min PBS washes before being mounted onto glass slides and coverslipped with Vectashield with DAPI. A 3 × 4 tiled z stack (7 optical sections comprising 35 μm total) image from both the left and right hemispheres of the BNST was obtained at 20× magnification using a Zeiss 800 upright confocal microscope. Scanning parameters and laser power were matched between groups. Images were preprocessed using stitching and maximum-intensity projection. The number of fluorescent cells in the dorsal and ventral aspects of the BNST were counted by a blinded scorer using the cell counter plug-in in FIJI (ImageJ). Each hemisphere was considered independently per mouse. One mouse in the flp-expressing group was a significant outlier for number of cells expressed in a ventral BNST hemisphere (ROUT, Q = 0.1%) and all data from that mouse were excluded. 3 male CRF-L10a reporter mice were injected with 200 nl of CTB 555 and CTB 647 bilaterally to the LH and VTA, respectively, as described above. 5 days following injection, mice were perfused as described above, the brains were extracted, and were stored in 4% paraformaldehyde for 24 h at 4 °C before being rinsed with PBS and transferred to 30% sucrose until the brains sank. 45 μm sections containing the BNST were collected as described above. Sections containing the BNST were mounted on glass slides and coverslipped using Vectashield. An image from the left and right hemispheres of a medial section of the BNST was obtained on a Zeiss 800 upright microscope using a 20×objective and 3 × 5 tiled z stacks (5 optical slices comprising 7 μm total). Images were preprocessed using stitching and maximum intensity projection, and were then analysed using the cell counter function in FIJI (ImageJ). Only cells positive for GFP (putative CRF neurons) were considered. Cells were scored exclusively as either 555+ only (LH-projecting), 647+ only (VTA-projecting), 555+ and 647+ (projecting to both LH and VTA), or 555− and 647− (unlabelled; neither LH- nor VTA- projecting). The total number of CRF neurons scored was calculated as the sum of all four groups, and percentages of each type were calculated from this value. Each hemisphere was scored and plotted independently (n = 6 images from 3 mice), and the dorsal and ventral BNST were considered separately. The average values were plotted as pie charts (Extended Data Fig. 5). For validation of 2C-cre line and comparison of CRF/2C mRNA cellular co-localization, mice were anesthetized using isoflurane, rapidly decapitated, and brains rapidly extracted. Immediately after removal, the brains were placed on a square of aluminium foil on dry ice to freeze. Brains were then placed in a −80°C freezer for no more than 1 week before slicing. 12 μm slices were made of the BNST on a Leica CM3050S cryostat (Germany) and placed directly on coverslips. FISH was performed using the Affymetrix ViewRNA 2-Plex Tissue Assay Kit with custom probes for CRF, 5-HT , and Cre designed by Affymetrix (Santa Clara, CA). Slides were coverslipped with SouthernBiotech DAPI Fluoromount-G. (Birmingham, AL). 3 × 5 tiled z stack (15 optical sections comprising 14 μm total) images of the entire 12 μm slice were obtained on a Zeiss 780 confocal microscope for assessment of CRF/2C colocalization. A single-plane 40× tiled image of a CRF/2C slice was obtained on a Zeiss 800 upright confocal microscope for the magnified image shown in Extended Data 6b, right. 3 × 5 tiled z stack (7 optical sections comprising 18 μm) images of 2C/Cre slices were obtained on a Zeiss 800 upright confocal microscope for the 2C/Cre validation. All images were preprocessed with stitching and maximum intensity projection. An image of the BNST from 3 mice in each condition was hand counted for each study using the cell counter plugin in FIJI (ImageJ). Cells were classified into three groups: probe 1+, probe 2+, or probe 1 and 2+. Only cells positive for a probe were considered. Results are plotted as average classified percentages across the three images. No specific method of randomization was used to assign groups. Animals were assigned to experimental groups so as to minimize the influence of other variables such as age or sex on the outcome. Pre-established criteria for excluding mice from behavioural analysis included (1) missed injections, (2) anomalies during behavioural testing, such as mice falling off the elevated plus maze, (3) damage to or loss of optical fibres, (4) statistical outliers, as determined by the Grubb’s test. A power analysis was used to determine the ideal sample size for behaviour experiments. Assuming a normal distribution, a 20% change in mean and 15% variation, we determined that we would need 8 mice per group. In some cases, mice were excluded due to missed injections or lost optical fibres resulting in fewer than 8 mice per group. For electrophysiology experiments, we aimed for 5–7 cells from 3–4 mice. Data are presented as means ± s.e.m. For comparisons with only two groups, P values were calculated using paired or unpaired t-tests as described in the figure legends. Comparisons across more than two groups were made using a one-way ANOVA, and a two-way ANOVA was used when there was more than one independent variable. A Bonferroni post-test was used following significance with an ANOVA. In cases in which ANOVA was used, the data met the assumptions of equality of variance and independence of cases. If the condition of equal variances was not met, Welch’s correction was used. Some of the sample groups were too small to detect normality (<8 samples) but parametric tests were used because nonparametric tests lack sufficient power to detect differences in small samples (Graphpad Statistics Guide – http://www.graphpad.com). The standard error of the mean is indicated by error bars for each group of data. Differences were considered significant at P values below 0.05. All data were analysed with GraphPad Prism software.
Agency: Cordis | Branch: FP7 | Program: CP-TP | Phase: HEALTH-2007-1.1-4 | Award Amount: 3.98M | Year: 2009
We are now entering an era of genomic exploration. The mouse is a pre-eminent model not only to map all mammalian genes, but to determine the function of each of these genes. There are now many large-scale projects to create collections of mutant and knock-out mice. However the next step which will add immense value to these collections will be the determination of the phenotype of each of these models. This will be an immense effort and will require input from laboratories and mouse phenotyping centres across the world. For the phenotype to be comparable across strains and between centres, it needs to be determined using standard protocols or SOPs, such as those contained in the European Mouse Phenotyping Resource of Standardised Screens (EMPReSS - www.empress.har.mrc.ac.uk). The goal of PhenoScale is to automate phenotyping in large-scale phenotyping using EMPReSS protocols; with the aim of adding economies of scale, enhancing automated data capture and increasing throughput. The technical development within the project is lead by the SME, TSE Systems GmbH, who will work with some of the leading mouse phenotyping centres in Europe at MRC, UK, CNR, Italy and GSF, Germany. The objectives are: 1) High-throughput Phenotyping: a) Development of a new high-throughput automated phenotyping platform PSS009 using home cage monitoring. 2) Development of new EMPReSS SOPs using PSS009 and validation of this approach with selected mouse models at different sites / European mouse clinics (cross-checking / cross-validation). 3) Optimization of the high-throughput technology systems by advanced interfacing and extended software capabilities. 4) Promotion of the new high-throughput systems and novel established SOPs to other phenotyping centres.