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Experimental studies on the melanopsin-mediated direct effects of light and sleep homeostasis in mice were realized through the use of Opn4 genetically ablated mice. The mutant mice were initially generated by Jackson laboratory. Gene disruption was made using a 38-bp fragment corresponding to a segment of the protein-coding region that was replaced by an IRES-lacZ reporter and neomycin resistance cassette (IRES-lacZ-neo). This mutation was designed to produce a loss-of-function mutation by deletion of amino acids 116 to 128, as well as insertion of the 6.9-kb reporter/resistance cassette. After electroporation into mouse embryonic stem cells of strain 129P2/OlaHsd, selecting those with G418 resistance, the cells were injected into blastocysts from a C57BL/6 strain. These chimeric mice were then crossbred with wild-type C57BL/6 producing the first generation heterozygotes. The mice were initially transferred from Stanford University, and the colony was extended at our facility in Strasbourg. Opn4-/- mice from two different genetic backgrounds (C57/Bl6J&129P2 and C57/Bl6 backcross) were studied during the course the doctoral program. The majority of the experiments were performed on Opn4-/-C57/Bl6&129P2 strain due to the availability of mixed genetic background mice at the start of the PhD. To control for genetic heterogeneity (C57/Bl6 and 129P2) the experiments were performed on male littermate homozygous offspring (Opn4+/+ & Opn4-/-) obtained from cross-breeding heterozygous (Opn4+/- x Opn4+/-) males and females. In addition, a second colony of melanopsin KO mice with a homogeneous C57/Bl6 genetic background was developed. The backcross began at Stanford University, and subsequently continued in Strasbourg, arriving at 10 backcross generations by the end of the PhD. At all times prior to experimentation mice were kept under environmentally stable conditions (12hL:12hD; 25 Â± 0.5Â° C, with food and water ad libitum.
4.1.2 The synaptotagmin10-Cre Bmal1 conditional KO mice (conditional deletion of the clock gene Bmal1 using the Syt10Cre driver)
The second transgenic mouse model included in the first experimental study was composed of Synaptotagmin10-Cre (Syt10Cre) Bmal1 conditional knock-out (Bmalfl/-) mice coming directly from the Max Planck Institute for Genetics in Göttingen, Germany. These mice were used specifically for transgenic modifications which invalidated their circadian clock in the suprachiasmatic nucleus. Here, we briefly describe the generation of this model; for details see Husse et al . Based on the Allen Brain Atlas (Lein et al., 2007) our collaborators from the Göttingen identified Synaptotagmin10 (Syt10) as a gene strongly expressed in the SCN with relatively few auxiliary expression sites in the CNS, even during development. The generation of a Syt10Cre driver line enables SCN targeting without targeting of peripheral, non-neuronal clocks. The exon 1 of the Syt10 gene was replaced by a Cre knock-in cassette, and after cloning of the Syt10Cre vector, the targeted clones were then injected into blastocysts of C57/Bl6 mice. These chimeric mice were then bred using wild-type C57/Bl6 to produce F1, followed by continued breeding to produce a colony by back-crossing to C57/Bl6. Syt10Cre were then crossed with Bmal1fl/fl to disable Bmal1 expression, solely in the SCN. Depending on the dosage of the Cre recombinase, mice with phenotypes ranging from minimal circadian perturbation to complete arrhythmicity were obtained, confirming the usefulness of the Syt10Cre driver line. These Syt10Cr/Cree Bmal1fl/- mice retained arrythmicity (locomotor activity, time course quantification of clock genes expression in the SCN) consistent with a disabled SCN. The controls used in this study were Syt10Cre/Cre Bmal1+/- mice that are rhythmic and allow us to control for Bmal1 and Syt10 gene expression (from our collaborators, data not shown). The genetic background of these mice was C57/Bl6J & C57/Bl6n, thus C57/Bl6J/N mice were used as an additional control for this study.
4.1.3 Arvicanthis Ansorgei
The Sudanian Grass Rat (arvicanthis ansorgei) is a diurnal rodent from the northern African grasslands. Though not extensively used in a laboratory setting, breeding facilities and previous research studies performed by the institute made this animal model available . The laboratory colony began in 1998 using trapped animals from southern Mali, initially 10 males and 15 females. To confirm the species was correctly identified, karyotipic analysis was performed on colony animals. Arvicanthis are closer in size to laboratory rats and as such are at least 5-10 fold higher in weight as compared to mice (150-250g vs. 25-35g). All animals were maintained in 12:12 light-dark (LD) cycle under constant temperature of 22 Â± 1 Â°C, with food and water ad libitum.
4.2 - Methods
4.2.1 PCR Genotyping
Genotyping for Opn4 mice was performed using a standard PCR protocol. Before PCR DNA concentration was determined using a standard spectrometer (BioRad). Primers used were:
Forward- Mel4: 5'- TCA TCA ACC TCG CAG TCA GC -3';
Reverse- Mel2: 5'- CAA AGA CAG CCC CGC AGA AG -3';
Forward-TodoNeo1: 5'-CCG CTT TTC TGG ATT CAT CGA C-3'.
Pcr consisted of 30 cycles: 95Â°C for 30s, 60Â°C for 30s, 72Â°C for 1 min. WT band was seen at 290 bp, mutant band at 950 bp.
Genotyping of Syt10 mice was performed using the following protocol: 38 cycles with an annealing temperature of 65ËšC using the primers-
Forward-5â€²-AGA CCT GGC AGC AGC GTC CGT TGG-3â€²;
Reverse-5â€²-AAG ATA AGC TCC AGC CAG GAA GTC-3â€²,
and for the KI Reverse-5â€²-GGC GAG GCA GG CCA GAT CTC CTG TG-3â€²
For WT, band was located at 426 bp, and for mutants, 538 bp. Bands were separated and run on a 1.5% agarose gel .
4.2.2 Protein quantification through western blot
Western blot procedures were used to determine amount of the melanopsin protein in mice retinas at different time points across the day. For all western blots a standard extraction protocol was used consisting of lysis buffer: 100 ÂµL (120 mg Tris Base 20mM, 435 mg NaCl 15mM, 500 ÂµL Triton 1%, 18.6 mg EDTA 1mM) and16 ÂµL protease inhibitor cocktail (Invitrogen). The lysates were then placed upon polyacrylamide gel for electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA). Reaction was then blocked with 5% skim milk and membranes were incubated overnight in a cold room at 4Ëš C with either: PA1-781 (1:1000, Affinity), ab65679 (1:4000, Abcam), UF006 (1:1000 Provencio), or D-18 (1:1000, Santa Cruz Biotechnologies), primary antibodies. Following this, the membranes were incubated with secondary antibodies, and then after exposition of the membranes to photoreactive film, the Opn4 protein was expected at ~53 and ~85 kDa (glycosylated form of the protein) via chemiluminescence (General Electric). Unfortunately after performing several experiments attempting to optimize the protocol conditions, the results were not convincing enough to pursue due to specificity/sensibility of the different antibodies and low level of expression of the protein in the tissue. Additionally, other teams have failed to obtain relevant and reproducible results from melanopsin western blot.
All surgical procedures were performed under deep anesthesia delivered intraperitoneally with Nembutal (68 mg/kg; University IRB-approved).
22.214.171.124 Lesion of the suprachiasmatic nuclei
Under pentobarbital anesthesia removal of the two SCNs was achieved using radio frequency lesions, according to published protocols . Lesions were achieved by heating the (250Âµm) tip of a Radionics (Burlington, MA) TCZ electrode to 55Â°C for 20 sec by passing RF current from a RFG-4 lesion generator (Radionics). Our group previously refined the various parameters to that we are able to create minimal lesions that spared surrounding brain structures. Briefly speaking, the lesions were performed stereotaxically (Kopf Instrument) with electrodes (0.3mm in diameter) introduced at the following coordinates (stereotaxic coordinates from zero ear bar, nose at +5Â°: lateral: +/-0.2 mm; antero-posterior: +3.4 and +3.6 mm; dorso-ventral: +0.95 mm; ) (Radionics Lesion Generator System) corresponding to the two SCN nuclei. Arrythmicity was confirmed by actimetry recordings under 12-12 LD cycle (10 days) and constant darkness (10 days), and effectiveness of lesions was assessed by periodogram analysis of locomotor activity (ClockLab, Actimetrics, Wilmette, IL, USA). Following the experiments lesions were verified histologically by performing Nissl staining on coronal brain sections.
126.96.36.199 EEG implantation
All animals underwent identical EEG implantation procedures, with the only exception being the size of the chip for arvicanthis. The chip used for these animals was more robust due to their increased size and activity as compared to mice.
Adult male mice and arvicanthis were implanted with a classical set of electrodes including two EEG, one reference, and two EMG electrodes at an age of 10-12 weeks at the time of surgery. Two gold-plated wires were inserted into the neck muscle tissue to record electromyogram (EMG) and two EEG electrodes were implanted on the dura skull over the right frontal and parietal cortex, respectively. In mice the electrodes were positioned in the frontal: 1.7mm lateral to midline, 1.5mm anterior to bregma, and parietal: 1.7mm lateral to midline, 1.0mm anterior to lambda.
In a subset of arvicanthis, EOG electrodes were implanted under the surface of the skin to record eye movements in order to characterize REM sleep. The five (or seven in case of EOG) electrodes were soldered to a connector and cemented to the skull. Each animal was housed in an individual cage with water and food provided ad libitum. After recovery from surgery (4-7 days) mice were connected to a swivel contact (6/12-Channel, Plastics One) through recording leads and allotted at least one week for cable adaptation. EEG, EMG and EOG signals were amplified, filtered, and their signals analog-to-digital converted and stored at 256 Hz (64-channel bipolar amplifier, Micromed France, SystemPLUS Evolution version1092).
All animals were given a 48-hour baseline assessment under 12h:12h light-dark conditions (150-200 lux- white fluorescent lights measured at the bottom of the cage using a lux meter). Following baseline, continuous sleep recordings were taken under a variety of experimental conditions. A minimum of 14 days under 12h:12h LD was used to habituate animals to their baseline condition before proceeding to the next recording condition. Prior to each experiment, a 24-hour period was assessed to ensure that the sleep wake distribution of the animals had returned to baseline levels. Sleep deprivations were performed by gentle handling .
4.2.4 Quantitative analysis of vigilance states
The behavioral states wakefulness (W), rapid eye movement sleep (REMS), and non-REM sleep (NREMS) were visually assigned for consecutive 4s epochs, either as Wake, NREM, or REM. In arvicanthis, EOG and a video capture system was used to verify the accuracy of the scoring with physiological behavior. All recordings were scored every 4-sec based on visual inspection of the EEG and EMG, as described previously and without knowledge of genetic background. If epochs contained signal artifacts they were included in the analysis for state amounts, yet excluded for power spectrum (see next). Amounts spent in each vigilant state were calculated in 5 and 30 min, and 1-, 12-, and 24h intervals.
4.2.5 Power spectrum analysis of the EEG
Once scoring was completed an average spectral profile was constructed using the entire experimental period, excluding epochs marked as artifacts. The EEG signal was subjected to Discrete-Fourier Transform (DFT) yielding power spectra between 0 and 128 Hz (0.25Hz resolution) using a rectangular calculation and an overlapping window of 4-sec. The frequency range 49-51 Hz was omitted from the analysis due to power-line artifacts in some of the recordings. For each state an EEG spectral profile were constructed by averaging all 4-s epochs scored as that state.
For all NREM sleep epochs, any time-dependent changes in EEG power for the delta (1-4Hz) band were examined. For epochs scored as wake, both theta (6-10 Hz) and gamma (40-70 Hz) bands were measured as EEG markers of alertness. To normalize for delta power during NREM sleep the last 4h of the (subjective) light period, the lowest period of homeostatic sleep pressure, was used and all values measured against it in mice. Spectral profiles of theta and gamma were calculated using an overlapping 10 min. window of 5-min. increments, yielding 13/hour, or for hourly values, were normalized against the lowest period of gamma and theta during the day (i.e. the least alert period). Custom programs were written for analysis in Pascal and then transformed into Visual C++.
4.2.6 Actimetric recordings
Locomotor activity recording was performed using either single-wheel cages or infrared motion capture, simultaneous or not to EEG recording. When the animals were in individual cages equipped with wheels (diameter of 22cm), locomotor activity was measured based on the number of wheel turns, recorded via a standard actimetry system (DataportDP24, Minimitter). Animals were recorded for at least 10 days for each of the light/dark exposure to establish clear patterns of activity. Infrared motion detectors were used for EEG sleep recording periods, as a wheel would be prohibitive due to the data cable attached to the animal. Following acquisition, data was organized into 10 5-minute bouts of locomotor activity using software (VitalView, Minimitter). Actograms and chi-squared analysis were performed using Clocklab (Actimetrics) following data transformation via Matlab.
4.3 - Anatomy
Once experimental protocols were completed, animals were deeply anesthetized with pentobarbital and perfused with heparin/NaCl followed by transcardial fixation for 15 minutes with 4% paraformaldehyde in PBS, pH 7.4 for in situ hybridization and immunohistochemistry. After dehydration in 30 % sucrose for 48 hours, the brains were frozen and cut in a cryostat.
To prepare tissues for immunohistochemistry or in-situ hybridization, samples were removed from -80Ëš C storage containers and placed in a standard cryostat at -18Ëš C (Leica). Slices were then placed in a free-floating buffer solution for later use. Immunohistological and in-situ hybridizations were performed by other members of the research team, Elisabeth Ruppert and Ludivine Choteau.