Supplementary Components1. enzyme glutamine synthetase (GS). Oligodendrocytespecific GS deletion will not impair myelination but disrupts neuronal glutamatergic transmitting, hence demonstrating a myelin-independent function for oligodendrocytes in supporting glutamate signaling in the brain. INTRODUCTION Glutamate is the major excitatory neurotransmitter in the brain. Following synaptic release, glutamate uptake and degradation are tightly regulated to achieve temporal and spatial signaling specificity and prevent cellular excitotoxicity (Kim et al., 2011; Sattler and Rothstein, 2006; Sheldon and Robinson, 2007). Currently, astrocytes are considered the sole glial cell type that contributes to glutamate uptake and degradation in the CNS (Jayakumar and Norenberg, 2016; Liang et al., 2006; Ortinski et al., 2010; Papageorgiou et al., 2018; Schousboe et al., 2013; Schousboe, 2019; Sun et al., 2017; Tani et al., 2014; Trabelsi et al., 2017; Yuan et al., 2017), as they express high levels of glutamate transporters and glutamine synthetase (GS), an enzyme that converts glutamate into glutamine. In keeping with this view, GS is frequently used as an astrocyte-specific marker (Armbruster et al., 2016; Habbas et al., 2015; Okuda et al., 2014; Papageorgiou et al., 2018; Theofilas et al., 2017; Tong et al., 2014). However, GS expression has also been reported in oligodendrocytes (Bernstein et al., 2014; Takasaki et al., 2010), glial cells known for producing myelin and ensheathing axons in the CNS (Pan and Chan, 2017). Although these results remain controversial (Anlauf and Derouiche, 2013; Jayakumar and Norenberg, 2016; Sun et al., 2017), it is of great physiological and clinical importance to identify all potential cellular and molecular components involved in the life cycle of glutamate. GS deletion from the brain results in neonatal death (He et al., 2010), and mutations in the GS gene produce severe neuropathology in humans (H?berle et al., 2012; Spodenkiewicz et al., 2016). Furthermore, glutamate dysregulation has been implicated in numerous pathological states, including epilepsy, stroke, and substance use disorders, as well as several neurodegenerative diseases (Jayakumar and Norenberg, 2016; Kalivas and Duffy, 1998; Reissner et al., 2015; Sheldon and Robinson, 2007; Spencer and Kalivas, 2017; van der Hel et al., 2005; Yuan et al., 2017). For these reasons, we sought to unequivocally determine whether GS is expressed by PF-4618433 oligodendrocytes and, if so, whether oligodendrocyte GS plays a role in maintaining glutamatergic synaptic transmission. RESULTS To assist in defining the cellular expression of GS, we immuno-stained brain sections from young adult (P60C75) mice, in which all astrocytes express EGFP (Zhang et al., 2014). Consistent with previous reports of astrocyte GS expression, there were numerous EGFP+ GS+ cells (Figure 1A, yellow arrows). However, we also observed an abundant population of GS+ EGFP? cells (Figure 1A, blue arrowheads; Figure 1E) in subcortical regions of the brain. Immunohistochemistry with aspartoacylase (ASPA), which is selectively expressed by mature oligodendrocytes (Larson et al., 2018), indicated that these GS+ EGFP? cells were oligodendrocytes (Figure 1B).Immunostaining in mice, where mature oligodendrocytes PF-4618433 communicate EGFP (Larson et al., 2018), also exposed wide-spread colocalization between EGFP+ oligodendrocytes and GS (Numbers 1C and ?and1F).1F). GS immunoreactivity was absent from cells expressing NeuN (neurons), CX3CR1 GFP (microglia), or PDGFR(oligodendrocyte progenitors) (Shape 1F; Numbers S1ACS1C). As yet another means of Rabbit polyclonal to IGF1R evaluating astrocyte and oligodendrocyte degrees of GS proteins, we utilized fluorescence-activated cell sorting (FACS) to isolate oligodendrocytes or astrocytes from the midbrain of and mice (Figure S1G), respectively, and quantified GS protein by western blot. Again, we detected GS expression in both astrocytes and oligodendrocytes (Figure 1G). Open in a separate window Figure 1. Oligodendrocytes Express GS mRNA and Protein(A) Confocal image PF-4618433 taken in the ventral midbrain of a P60 mouse, immunolabeled with anti-GS. Yellow arrows indicate EGFP+ GS+ astrocytes; blue arrowheads indicate EGFP?GS+ cells. (B) Immunostaining for the mature oligodendrocyte marker aspartoacylase (ASPA) and GS in the ventral midbrain of a P60 wild-type mouse; blue arrowheads indicate GS+ oligodendrocytes. (C) Immunostaining for GS in the ventral midbrain of a P60 mouse. (D) hybridization for and in the ventral midbrain of a P63 wild-type mouse. In (A)C(D), underneath panel is a focus from the particular area inside the red box in the very best image. (E) Quantification from the percentage of GS+ cells that are EGFP+ or EGFP?; n = 3 mice. (F) Quantification of % oligodendrocytes (MOBP EGFP+), neurons (NeuN+), microglia (CX3CR1 GFP+), or oligodendrocyte progenitors (PDGFRa+) expressing GS; n = 3C4 mice per cell type. For instance images, see Numbers S1ACS1C. (G) Traditional western blot for GS in FACS-isolated oligodendrocytes and astrocytes. The cellular number.