Fig 2: Biochemical fractionation and WB procedure.(a) Schematic diagram of a coronal mouse brain section indicating the region removed using biopsy punch pen. Modified from Allen Brain Atlas (http://atlas.brain-map.org/#atlas=1&plate=100960236&structure=549&x=5280&y=3744.0000697544647&zoom=3&resolution=11.97&z=5). (b) Flow chart to illustrate fractionation methodology. TxP = Triton X-100 insoluble synaptic fraction (pellet of last spin), TxS = Trinton X-100 soluble extrasynaptic membrane fraction (supernatant of last spin). (c) Representative WB composite images showing bands corresponding to markers, Pan-Cadherin and PSD-95 in different fractions. S1 = supernatant 1 - total lysate; S2 = supernatant 2 - cytosol; TxS = Triton X-100 soluble supernatant – extrasynaptic membrane; TxP = Triton X-100 insoluble pellet – synaptic membrane.

Fig 3: Distribution of iGluSnFR at the neuronal membrane.(A) Experimental paradigm and timeline for sparse transfection and subsequent imaging of iGluSnFR. (B) Representative image showing the density distribution of a density analysis of iGluSnFR probes at the postsynaptic membrane of an iGluSnFR transfected neuron, where areas in the red gradient have a denser distribution of probes and blue colored regions represents less dense regions. (C) Representative image of two synaptic boutons that colocalize to PSD95, showing that there is increased density of iGluSnFR probes closer to the center of the cluster. (D) Pair correlation of iGluSnFR clusters that colocalize to PSD95, demonstrating increasing clustering near the center of the synapse (n = 30 synapses). Figure 1—source data 1.Source data for Figure 1.

Fig 4: Increased expression of D1 dopamine receptors and hyperactivated striatal D1-pathway in MPS-IIIA mice.a A representative scheme of a striatal dopaminergic synapse, including D1-direct and D2-indirect DA signalling, on the 3',5'-cyclic adenosine monophosphate (cAMP) intracellular pathway. b In the striatum, MPS-IIIA mice showed increased expression of TH, evaluated by WB analysis. c MPS-IIIA mice have increased striatal DA, d increased expression of D1Rs and d’ no change in the expression of D2Rs. e These data were confirmed by immunofluorescence analysis against D1R. Representative images of D1Rs/DAPI immunofluorescence of the shell of accumbens nucleus (nAcc), the dorsolateral (DLS) and dorsomedial (DMS) striatal areas of WT and MPS-IIIA mice are shown (scale bar: 20 µm). e' Triple immunolabeling against D2R, SYN and PSD-95 revealed decreased colocalization of D2R/SYN and increased colocalization of D2R/PSD-95, suggesting decreased and increased expression of D2R at pre- and post-synaptic level, respectively. Representative images of D2R/SYN/PSD-95 immunofluorescence of WT and MPS-IIIA mice are shown. Digital zoom of the boxed area showing coexpression of D2R/SYN (arrows for WT mice, and arrowheads for MPS-IIIA mice) and of D2R/PSD-95 (arrows for MPS-IIIA mice), (scale bar: 50 µm). f, f' MPS-IIIA showed hyperactivation of DARPP-32 intracellular signalling, which is rescued by SCH-23390, but not by haloperidol, treatment. Representative WB for each condition is presented. Dashed lines in (d') delineate cut parts from the same western blot. Histograms represent mean ± S.E.M. *p < 0.05, **p < 0.01 vs WT, between groups; #p < 0.05 vs MPS-IIIA veh, within group.

Fig 5: Met colocalizes with gephyrin and PSD95. Slices prepared from the dentate gyrus or hippocampal neurons were stained for the nuclear stain Hoechst, Met, gephyrin and PSD95 and submitted to confocal fluorescence microscopy. For overview, images of hippocampal neurons (A,B) or the granule cell layer of dentate gyrus (C,D) were stained with antibodies for MAP2 and Hoechst. Scale bar 20 µm. Insets in panels (A–D) are magnified in (A’–A”’,B’–B”’,C’–C”’,D’–D”’). Scale bar 10 µm. (A’–D’): Met staining, (A”–D”): gephyrin or PSD95 staining as indicated, (A”’–D”’): merged images of the respective representations in (A’–A”,B’–B”,C’–C”,D’–D”). Arrow heads indicate colocalizing spots at the corresponding locations. (E) 20 µm3 cubes representative of the granule cell layer (GC layer), proximal (proximal ML) and distal molecular layer (distal ML) of the dentate gyrus were analyzed for the percentage of gephyrin clusters colocalizing with Met. n = 11 for all groups. Error bars: SEM.