Presence of Cr in SVs from the mouse brain.

(A) Representative raw traces from CE-MS of indicated molecules from samples immunoisolated by the control IgG (blue) at 0-2 °C, the monoclonal anti-Syp antibody at 0-2 °C (red) and the anti-Syp antibody at room temperature (RT, green). Q1/Q3 for identifying targets were indicated. (B-G) Quantification of the amounts of indicated molecules. The amount of a molecule was divided by the amount of the anti-Syp antibody bound to magnetic beads. Note Glu (B), GABA(C), ACh (D), 5-HT (E), Cr (F), but not alanine (G) was higher in SVs pulled down by the anti-Syp antibody at 0-2 °C than those pulled down by the IgG control or those pulled down at the RT. n=10 (B-E, and G) or 14(F) samples per group, ***, p<0.001, ns, not significant. one-way ANOVA with Tukey’s correction.

SLC6A8 and Cr in SVs.

(A) A schematic illustration of the strategy for generating SLC6A8 knockouts using CRISPR/Cas9. An iCreERT2-WPRE-pA cassette (∼3.5 kb) was inserted immediately downstream of ATG in the SLC6A8 gene, substituting bp 4 to bp 51 in exon 1 (E1). (B) Representative raw traces of Creatine immunoisolated by control IgG from WT mice (blue), the anti-Syp antibody from WT mice (red), IgG from SLC6A8 KO mice (blue) and the anti-Syp antibody from SLC6A8 KO mice (red). (C-H) Quantification of indicated molecules. Note the selective reduction of Cr in SVs from SLC6A8 KO mice. n=14 samples per group. ***, p<0.001, ns, not significant. one-way ANOVA with Tukey’s correction.

Reduction of SV Cr in AGAT knockout mice.

(A) Representative raw traces of Creatine, pulled down by IgG or anti-Syp from AGAT+/+, AGAT+/- and AGAT-/- mice. (B-G) Quantification of indicated molecules. Cr was significantly decreased in AGAT-/- mice as compared to Cr in AGAT+/+ or AGAT+/- mice (B). GABA was significantly decreased in SVs from AGAT-/- mice as compared to AGAT+/+ mice (D) but the difference was smaller than that of Cr. Glu (C), ACh (E), 5-HT (F) and alanine was not different among AGAT+/+, AGAT+/- and AGAT-/- mice. n=10 samples per group. ***, p<0.001, ns, not significant. one-way ANOVA with Tukey’s correction.

Expression pattern of SLC6a8.

(A) A diagram illustrating the generation of SLC6A8-HA KI mice. 3*HA, T2A and CreERT2 were inserted in-frame, before the stop codon, to the C terminus of SLC6A8 protein. (B-K) Low magnification photomicrographs of coronal sections immunohistochemically labelled with the anti-HA antibody in SLC6A8-HA mice. (I and M) immunostaining with the anti-HA antibody in control WT mice. Abbreviations: Pir, piriform cortex; M, motor cortex; LS: lateral septum; Hp: hippocampus; Hb, habenular nucleus; Vp: ventral posterior nucleus of thalamus; Au auditory cortex; Amyg: amygdala; Ip, interpeduncular nucleus; Pn; pontine nucleus; Cb, cerebellum; Pr, prepositus; SC, Superior colliculus; MVe, medial vestibular nucleus. Scale bar: 500 μm.

Cr release in brain slices from WT and SLC6a8 knockout mice.

(A) Neuronal depolarization induced by 70 mM K+ and time points for collecting the release sample. “Control” samples were collected 1.5 min to 0.5 min before K+ stimulation, “70 mM K+” ACSF samples were collected during 70 mM K+ stimulation, and “wash” samples were collected 10 mins after washout with ACSF. Efflux of Glu (B), GABA (C) or Cr (D) from WT or SLC6A8 KO male mice (n=7 samples per group). Note that a small amount of Cr released in SLC6A8 KO mice did not return to the baseline after 10 mins of washing. (E-G) Ca2+ dependent release of Glu, GABA and Cr in WT and SLC6A8 KO mice (n=5 samples per group). ***, p<0.001, ns, not significant. one-way ANOVA with Tukey’s correction. #, p<0.05, ##, p<0.01, paired t test.

Cr release in WT and AGAT knockout mice.

(A) A schematic diagram illustrating the strategy of AGAT knockout first. With the AGAT gene (also known as GATM) shown in the upper part, and the gene targeting strategy in the lower part. The homologous arm is approximately 10 kb. A targeting cassette, containing Frt-flanked lacZ and neomycin, was inserted downstream of exon 2. At the same time, exon 3 of AGAT was flanked by two loxP sites. K+ induced release of Glu (C) and GABA (D) were not significantly different among AGAT+/+, AGAT+/- and AGAT-/- mice, whereas that of Cr (B) was significantly lower in AGAT-/- mice than those in AGAT+/+ and AGAT+/- mice.

Inhibitory effects of Cr on cortical neurons.

(A) A photograph showing recording at layer 4 in the somatosensory cortex. Scale bar: 10 μm. (B) Patch-clamp recording of a pyramidal neuron. Scale bar: 10 μm. (C) Ratios of Cr - responsive and -unresponsive neurons in the region. (D) Representative raw electrophysiological traces showing inhibition of evoked firing by Cr, with the lower panel showing the stimulus protocol. (E) Evoked spike numbers in response to different current injections from (D). (F) Relationship between evoked spike numbers and different current injections to neurons that were inhibited by Cr (n=16). (G) The same for Cr-unresponsive neurons (n=35). (H) Rheobase for Cr responsive neurons. (I) Evoked spike number when these neurons were injected with current of rheobase + 50 pA. (J) input resistance. *, p<0.05; **, p<0.01; ***, p<0.001, paired t test.

Cr uptake into synaptosomes and SVs.

(A) Markers for subcellular organelles detected in synaptosomes prepared from WT mice or mice with Slc6a8 gene fused in frame to the HA epitope. SLC6A8-HA was enriched in synaptosomes (Sy or 4-Sy) or crude synaptosomes (P2) (see methods), as were other markers for the subcellular organelles in synaptosomes but not the marker for myelin (MBP). (B) Representative electron micrographs and histograms of size distribution in synaptosomes isolated from WT (Slc6a8+/Y) and Slc6a8 KO (Slc6a8-/Y) mice by Ficoll density-gradient centrifugation. Sy, synaptosome; SV, synaptic vesicles; Mt, mitochondria; PSD, post-synaptic density. Bar, 20 m. (C) Cr uptake into synaptosomes (n=5 per group). The two left columns were results from WT mice and the two right columns from Slc6a8 knockout mice. The control baseline was [14C]-Cr uptake at 0 °C at 10 mins (205). Cr uptake into synaptosomes at 37 °C measured at 10 mins was observed in WT synaptosomes. Uptake into Slc6a8 knockout synaptosomes was significantly reduced when compared to the WT synaptosomes. ***, p<0.001, one-way ANOVA with Tukey’s correction. (D) uptake of [13C]-Cr into immunoisolated SVs in the presence or absence of ATP. (n=3 samples per group)***, p<0.001, t test.