Amyotrophic lateral sclerosis (ALS) is an incurable, progressive and fatal neurological disorder. Early stage detection and prediction of disease progression is at present extremely difficult. Currently, one of the few available non-invasive imaging techniques to probe ALS pathophysiology is proton spectroscopic imaging¹. The disease is manifested by motor neuron degeneration, but recent evidence suggests that glutamate excitotoxicity and glial cell activation play an important role in early development of ALS pathology². In this study, we report on using a glutamate-sensitive CEST approach³ and manganese-enhanced MRI as novel biomarkers for a better understanding of ALS pathophysiology at different stages of the disease.Five male SOD1G93A transgenic ALS mice (B6SJL-Tg(SOD1*G93A)1Gur/J) and 5 male normal wild type (WT) littermates (Jacksons Laboratory, Bar Harbor, ME) were used in this study. MRI was performed using a horizontal bore Bruker Biospin 11.7T scanner Bruker, Ettlingen, Germany). A 72 mm volume resonator and a 4x1 phased array spinal cord coil were used for transmission and image acquisition, respectively. For glutamate-sensitive CEST of the spinal cord, we used an on-resonance variable delay multi-pulse sequence4 that is sensitive to fast exchanging protons and therefore detects predominantly glutamate in neuronal tissue. The VDMP sequence used 32 pulse-exchange modules with a 2 ms pulse width and a B1=46.8 μT was used. Ten mixing times (0 to 150 ms) were used for the MTC and GluCEST quantification by fitting the onVDMP buildup curves. For manganese-enhanced MRI (MEMRI), a spin echo T1-weighted pulse sequence with a TR/TE=550/8 ms, number of averages=8, FOV=12.8x12.8 mm, a matrix=192x192, number of slices=20, and a slice thickness=1 mm was used (spatial resolution of 67 μm in both dimensions). Animals were imaged pre- and 24 hours post-MnCl₂ i.p. injection (0.4 mmol/kg bw). All animals were imaged at the pre- and post-symptomatic stage of the disease (67 and 125 days, respectively). Lumbar spinal cord sections were stained with anti-GFAP antibody as marker for astrocyte activation. For in vitro MEMRI cell activation studies, glial restricted progenitor (GRP)-derived astrocytes, undifferentiated GRPs, and fibroblasts were treated with and without a cytokine activation cocktail and with and without 2 μM of the voltage-gated Ca²⁺ channel blocker verapamil. Cultures were then incubated with 0.4 mM MnCl₂ for 30 min. A total of 5x105 cells (n=2) were imaged for each cell incubation paradigm. Data were analyzed using PROC MIXED (Statistical Analysis System), employed with Type III fixed effects to evaluate the significance and lowest mean squares for direct comparisons between groups. Fast-exchange sensitized VDMP MRI of the lumbar spinal cord showed a significantly higher signal in the grey matter of ALS spinal cord compared to wild type controls (p< 0.05) at only at the pre-symptomatic stage of the disease (Fig. 1). Based on exchange rate considerations, we interpreted this to be due predominantly to an increased level of glutamate. MEMRI demonstrated a significantly higher contrast in both white and grey matter of ALS mice at the post-symptomatic stage, which corresponded to anti-GFAP immunostaining for activated astrocytes (Fig. 2). In vitro manganese cellular uptake studies showed a significant increase of T1-w signal only in activated astrocytes that was blocked by the addition of verapamil indicating calcium channels as mechanism of Mn²⁺ intracellular transport.Increased fast-exchange sensitized VDMP signal intensity during the pre-symptomatic stage of the disease may be used to predict if, when, and where ALS pathology develops. Increased MEMRI signal during the post-symptomatic stage of ALS corresponds to the histological presence of activated astrocytes, which are known to exhibit changes in calcium metabolism⁵. This agreed with the in vitro MEMRI experiments where activated astrocytes without Ca²⁺ blocker showed the highest signal. The two MRI modalities open up new avenues to study glial cell activation and ALS pathophysiology, and may be further used in drug development aimed to reduce glutamate excitoxicity.