Mice Brain Slices (40-100µm) cut with 5100mz Vibrotome for Immunofluorescence

Abstract

(from Functional consequences of postnatal interventions in a mouse model of Fragile X syndrome, Rais et al, 2021)

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Background

Fragile X syndrome (FXS) is a leading genetic cause of autism and intellectual disability with cortical hyperexcitability and sensory hypersensitivity attributed to loss and hypofunction of inhibitory parvalbumin-expressing (PV) cells. Our studies provide novel insights into the role of excitatory neurons in abnormal development of PV cells during a postnatal period of inhibitory circuit refinement.

Methods

To achieve Fragile X mental retardation gene (Fmr1) deletion and re-expression in excitatory neurons during the postnatal day (P)14-P21 period, we generated CreCaMKIIa/Fmr1Flox/y (cOFF) and CreCaMKIIa/Fmr1FloxNeo/y (cON) mice, respectively. Cortical phenotypes were evaluated in adult mice using biochemical, cellular, clinically relevant electroencephalogram (EEG) and behavioral tests.

Results

We found that similar to global Fmr1 KO mice, the density of PV-expressing cells, their activation, and sound-evoked gamma synchronization were impaired in cOFF mice, but the phenotypes were improved in cON mice. cOFF mice also showed enhanced cortical gelatinase activity and baseline EEG gamma power, which were reduced in cON mice. In addition, TrkB phosphorylation and PV levels were lower in cOFF mice, which also showed increased locomotor activity and anxiety-like behaviors. Remarkably, when FMRP levels were restored in only excitatory neurons during the P14-P21 period, TrkB phosphorylation and mouse behaviors were also improved.

Conclusions

These results indicate that postnatal deletion or re-expression of FMRP in excitatory neurons is sufficient to elicit or ameliorate structural and functional cortical deficits, and abnormal behaviors in mice, informing future studies about appropriate treatment windows and providing fundamental insights into the cellular mechanisms of cortical circuit dysfunction in FXS.

Method

Immunofluorescence

Age-matched P14, P21, and P60-70 male Ctrl WT, cOFF, Ctrl KO and cON mice were euthanized with isoflurane and sodium pentobarbital and perfused transcardially first with cold phosphate-buffered saline (PBS, 0.1 M) to clear out the blood and then with 4% paraformaldehyde (PFA) in 0.1 M PBS for fixation. Brains were removed and post-fixed for 2–4 h in 4% PFA. 40-100 μm brain slices were obtained using a vibratome (5100mz Campden Instruments). AuC was identified using hippocampal landmarks and the Franklin and Paxinos mouse brain atlas (Paxinos and Franklin, 2004). For each brain, an average of 5–6 brain slices containing AuC were collected.