Inova

The 6- and 12-month-old mice (n = 5–7) underwent MRI/MRS scanning to evaluate genotype- and treatment-induced differences in brain metabolites of FC and HIPP. MRI/MRS analyses were conducted at 4.7 T on a Varian/Agilent Inova horizontal bore system (Agilent, Palo Alto, USA) using a combination of volume and surface coil (RAPID Biomedical, Rimpar, Germany) according to a protocol described in Scuderi et al. (2018 (link)). Briefly, mice were anesthetized with isoflurane (IsoFlo, Abbott SpA, Berkshire, UK) 1.5–2.5% in O₂ 1 L/min. Anatomical T2-weighted sagittal MRIs were acquired for the positioning of the voxels for MRS. Localized 1H-MRS (PRESS TR/TE = 4,000/23 ms) were collected from HIPP and FC (volume 9.5 and 9.1 μl, respectively) as shown in Figure 4A, according to a quantitative protocol (Canese et al., 2012 (link)).
The following six metabolites were considered: N-acetyl-aspartate (NAA), myo-inositol (mINS), the sum of creatine and phosphocreatine (Cr + PCr), glutamate (Glu), glutamine (Gln), and total choline (tCho). Metabolite concentrations are expressed in mmol/L (mM). Moreover, we evaluated the NAA/Cr ratio and mINS/Cr.

Following dissolution, 2 ml of hyperpolarized δ-[1-¹³C]gluconolactone was rapidly transferred to a horizontal 3 T scanner (BioSpec 105 mm bore diameter, Bruker) to evaluate T₁ (n = 3, TR = 3 s/FA = 10°) or to a vertical 11.7 T MR system (INOVA, Agilent Technologies) to evaluate percent polarization (n = 3, TR = 300 s/FA = 90°/NA = 16) and T₁ (n = 3, TR = 3 s/FA = 13°). Spectra were processed by peak integration using MestreNova (v12.0.4, Mestrelab, Spain). For determination of T₁, the quantified peak integrals were corrected for flip angle and fitted with a mono-exponential curve. The liquid-state polarization and SNR improvement were evaluated by comparing the first hyperpolarized spectrum of the dynamic set to the corresponding thermal equilibrium spectrum after correction for flip angle, number of averages and back calculating the value to the time of dissolution (18 to 25 s prior to first spectra acqusition).

After the first boost, a group of mice (batch I, n = 7 per group; see Fig. 2) between 8 and 9 weeks of age underwent MRS analyses, with the aim of measuring the concentration of biochemical parameters in prefrontal cortex and striatum (see also37 (link)). All experiments were conducted on a 4.7 T Varian Inova animal system (Varian/Agilent Inc. Palo Alto, CA, USA), equipped with actively shielded gradient system (max 200 mT/m, 12 cm bore size) and a combination of volume coil for transmission and a surface coil as receiver (Rapid Biomedical, Rimpar, Germany). Further details on the apparatus are described in38 (link). Single voxel localised ¹H MR spectra (PRESS, TR/TE = 4000/23 ms, ns = 256 or 512) were collected from relevant brain areas: prefrontal cortex (PFC, 9.7 μl) and striatum (STR, 9.7 μl) (see Fig. 2). Quantitative MRS protocol, including water T2 measurements, was applied, see39 (link) for details.
Spectra were analysed using LCModel40 (link) that calculates the best fit to the experimental spectrum as a linear combination of model spectra (spectra of metabolite solutions). Further details on the methodology are described in Supplementary Information and in38 (link).

Magnetic resonance imaging was done on the 4.7 Tesla Varian Inova scanner at the Peter S. Allen MR Research Centre at the University of Alberta. We acquired blood oxygenation level dependent (BOLD) fMRI images with a T2^*-weighted echo planar imaging sequence using these parameters: volume time 2.0 s, single shot, repeat time 2.0 s, echo time 19.0 ms, 3.0 mm isotropic voxels, 80 × 80 matrix, 240 × 240 mm² field of view, 3.0 mm slice thickness, 36 axial slices, 108 mm through-plane coverage, interleaved slice collection order. We used 80% partial k-space in the phase encode direction (anterior-posterior). The fMRI scanning volume covered the entire cerebral cortex except for the ventral-posterior tip of occipital cortex in participants with larger heads. A high resolution T1-weighted structural scan was also acquired for each participant. This scan utilized a magnetization-prepared rapid acquisition gradient echo (MPRAGE) sequence with parameters: TR 9.4 ms, inversion time 300.0 ms, relaxation delay time (after readout prior to inversion) 300.0 ms, linear phase encoding, TE 3.7 ms, matrix 240 × 192 × 128, field of view 240 × 192 × 192 mm³, 1.0 × 1.0 × 1.5 mm³ voxels, whole brain coverage.

Lyophylized tissue powder was extracted three times with water, and reduced to dryness. This dry, water-soluble fraction was reconstituted in 600 μL sodium phosphate buffer (50 mM, pH 7). Seventy microliters D₂O and 30 μL of a standard buffer solution [3.73 mM DSS (disodium 2,2-dimethyl-2-silapentane-5-sulfonate), 0.47 % (w/v) sodium azide] were added. Samples were vortexed for 1 min, sonicated for 30 min, and transferred to a standard Shigemi microcell NMR tube. All ¹H-NMR spectra were acquired on a 500 MHz Inova (Varian Inc., Palo Alto, California) spectrometer equipped with a 5-mm HCN Z-gradient pulsed-field gradient (PFG) cold probe. Data was collected at 25 °C using the first transient of the NOESY-presaturation pulse sequence, which was chosen for its high degree of quantitative accuracy. Spectra were collected with 256 transients using an 8-second acquisition time and a 1-second recycle delay.