Immunoisolation and culture of CNS neurons from postnatal mice

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Goal of the study

To decipher the role of glial LSR in the cholesterol crosstalk within the CNS, we used a conditional Cre/lox recombination system developed in a glia-specific transgenic mouse line that allow for temporally controlled site-specific recombination (Slezak et al. 2007). Their approach depends on cell specific expression of TAM-dependent CreERT2 recombinase and on transgenes derived from bacterial artificial chromosomes (BACs). The Cre mouse line showed highest Cre-mediated recombination in the glia cells from cerebellum, hippocampus, and olfactory bulb with lower activity in other brain areas and eye. Outside the CNS, Cre activity was observed in both the spleen and skin (Slezak et al. 2007). In the present study, we suppressed LSR expression in glial cells at the age of 2 months using the TAM-induced GlastCreERT2 floxed lsr (cKO) mouse model, followed by a series of behavioral studies for the assessment of activity, olfaction, vision, sociability, and short- and long-term memory. The behavioral phenotyping revealed a series of traits resembling AD, with a sequence of events mimicking early and later steps of the pathology. Our findings therefore demonstrate that glial LSR disruption is enough to switch from normal to pathological aging of the CNS.

Materials and methods


Animal studies were conducted in accordance with the European Communities Council Directive (EU 2010/63) for the use and care of laboratory animals. All experimental procedures were carried out in accordance with the ethical committee CELMEA N°066 with an approval number APAFIS #12079-201711081110404. Animals were housed in certified animal facilities (#B54-547-24) on an inverted 12-hour light/dark cycle with a mean temperature of 21–22 ◦C and relative humidity of 50 ± 20 %, and provided a standard chow diet (Envigo Teklad, Gannat, France) and water ad libitum. To generate conditional knockout male mice (cKO, n = 18), Cre mice were crossed with LSRloxP/loxP.

Breeding and generation of experimental groups

For cKO generation, we first crossed Cre mice (ICS, Ilkrich, France) with floxed lsr mice LSRloxP/loxP mice together to obtain the first generation F1 (Figure 3.1). The F1 generation was about 50 % heterozygous for the lox P allele and hemizygous for the Cre transgene (GLASTCreERT2-LSRloxP/wt). The GLASTCreERT2-LSRloxP/wt mice were crossed back to the LSRloxP/loxP mice and approximately 25 % of the F2 generation contained cKO mice; the experimental mice. Since, we need a sufficient number of mice for statistical robustness (n = 18), those 25 % of F2 were crossed together (avoiding close relatives mating) in order to increase their number (Figure 3.1). Concerning control groups, we chose to maintain two control groups, wild type mice (WT- negative controls,n = 20) and Cre (n = 18). Both Cre and cKO were induced with TAM in order to link any behavioral/physiological change to the loss of LSR function (Figure 3.3). To avoid “litter effect’’ i.e. the tendency for littermates all to respond in the same way to stimuli, unlike animals from different litters, each mouse used for behavioral phenotyping came from a different mother. All 18 cKO mice were a result of 18 different breading pairs.
Figure 3.1: Breeding plan. (A) LSRfl/fl crossed with Cre mice line to obtain 50% GLASTCreERT2-LSRloxP/wt in F1. (B) The GLASTCreERT2-LSRloxP/wt mice were crossed against LSRloxP/loxP mice to obtain 25% cKO in F2.

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Genotyping of Cre and floxed lsr mice

Mouse genomic DNA was isolated from ear biopsies following overnight digestion at 55 °C in Direct PCR lysis buffer (Viagen Biotech, Los Angeles, CA, USA) and 0.2 mg/mL of Proteinase K (Invitrogen, Cergy Pontoise, France) followed by heat inactivation of Proteinase K. For genotyping of Cre expression, two sets of primers were used for the same PCR reaction, TK139 and TK140, which gives a 350 base pair (bp) DNA band, specific for the Cre allele, and ADV28 and ADV30, a 250 bp band, which correspond to a myogene, that serves to verify the success of the PCR amplification. The PCR reaction temperature were as follows: 95 °C for 2 minutes, 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 30 seconds for 30 cycles, then 72 °C for 10 minutes. In order to detect whether the mice were heterozygous or homozygous for the LSRloxP/loxP, the protocol of ICS (# IR00004190 / K4190) was followed, using two sets of primers; he mice were put to fast 6-7-hours before the test time, with free access to water. The test was carried out in the room of the tested mice, in red light, and in their own cages. A barrier was put in the middle of the cage, and the cookie was buried 1 cm under the litter, in the opposite side of the cage. The test was filmed using a video camera in night mode, the barrier was removed and the latency to find and start eating the cookie was measured (Figure 3.6).

Table of contents :

Chapter II (Article I)
Short resume:
1. Introduction
2. Materials and methods
2.1. Animals
2.2. Immunoblots
2.3. RNA extraction and RT-qPCR
2.4. Mixed cell culture
2.5. Immunoisolation and culture of CNS neurons from postnatal mice
2.6. Immunocytochemistry
2.7. Statistical analysis
3. Results
3.1. Lsr RNA profiling in mice brain
3.2. LSR differential expression in different brain regions and the retina
3.1. LSR expression in neurons and glial cells
3.2. Glial cells highly express LSR in cerebellum
4. Conclusions and discussion
5. Additional data and supporting information
Chapter III
Short resume
1. Introduction
1.1. Scientific question
1.2. Goal of the study
2. Materials and methods
2.1. Animals
2.2. Breeding and generation of experimental groups
2.3. Genotyping of Cre and floxed lsr mice
2.4. Tamoxifen (TAM) induction of GlastCreERT2 enzyme
2.5. Behavioral tests
2.6. RNA extraction and RT-qPCR
2.7. Blood glucose test
2.8. Statistical analysis
3. Results and conclusions
3.1. Lsr downregulation in cKO mice
3.2. Activity and anxiety
3.3. Olfaction
3.4. Memory
4. Discussion and perspectives
5. Supplementary data
6. Preliminary results
6.1. Lsr excision from hippocampus, cerebellum, and olfactory bulb
6.2. Effects of glia-specific lsr excision on cholesterol metabolism
Chapter IV
Short resume:
1. Introduction
2. Results
2.1. Home cage activity:
2.2. Free exploratory paradigm:
2.3. Open field test:
2.4. Buried cookie test:
2.5. Odor discrimination test:
2.6. Object recognition test:
2.7. Three-chambered sociability test:
2.8. Y-maze:
2.9. Barnes maze:
3. Conclusion and discussion
General discussion


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