Fig 2: Synaptic neurotransmitter release probability diversity impacts long-term synaptic plasticity triggered by Poisson spike input series.(A) Illustration of the model used in the simulation. Two leaky integrate-and-fire neurons with a synaptic connection received independent Poisson spike trains. Similarly to Figure 3, the release probability of neuron 1 [subject to alteration by the neighboring astrocyte(s)] is modelled by three different gamma distributions representing control, AP5, and AP5-with-control mean conditions. (B) Summary of simulation. Major vertical axis (left, blue) shows the relative differences in the final synaptic weights in AP5 (left plot) or AP5-with-control mean (right plot) compared to the control condition. The relative differences (blue dots) are plotted against the final synaptic weight change in control condition (horizontal axis). The binned data are shown as green bars (minor vertical axis: right, green). The plots indicate that spike inputs that promote potentiation under control condition tend to favor weaker potentiation in either AP5 or AP5-with-control mean, and spike inputs that promote depression under control condition tend to favor weaker depression in either AP5 or AP5-with-control mean. (C) The reduction of long-term plasticity in AP5 and AP5-with-control mean representing situations of narrowed release probability variance, relative to the control condition. The reduction of plasticity is defined by the mean of the differences between the final weights in NMDAR-blocked conditions and the control condition with a sign correction since long-term potentiation and long-term depression change weights in opposite directions.

Fig 3: Deletion of LAR-RPTPs in vivo selectively decreases the NMDAR-/AMPAR-EPSC ratio at Schaffer collateral synapses in the hippocampus without affecting AMPAR-mediated synaptic connectivity.(A) Schematic of experimental strategy for panels B-E (left), and representative images of injection sites in the CA3 region of the hippocampus (right; scale bar = 0.5 mm). The CA3 region of newborn littermate LAR-RPTP triple cKO mice was stereotactically infected with AAVs expressing ?Cre (as a control) or Cre, and Schaffer-collateral EPSCs elicited by electrical stimulation were recorded from CA1 neurons at P30-37. (B) AMPAR-mediated unitary EPSCs elicited by electrical stimulation of Schaffer collaterals in the presence of 3 mM Sr2+ extracellularly (0 Ca2+) are not altered by the deletion of LAR-RPTPs. EPSC peak amplitude (showed by arrows) was analyzed within 50–500 ms after the stimulation (dotted box). Results confirm that removal of LAR-RPTPs does not affect post-synaptic AMPAR amplitudes. (C) Input-output curves of AMPAR-EPSCs confirm that AMPAR-mediated synaptic connectivity is not affected by deletion of LAR-RPTPs from CA3 neurons. (D) Presynaptic LAR-RPTP deletion suppresses the NMDA/AMPA ratio (left, representative traces; right, summary graph). The peak amplitudes of AMPAR-EPSCs were recorded at -70 mV in PTX, and composite AMPAR- and NMDAR-EPSCs were then recorded at +40 mV in the same cells, with the NMDAR-EPSC component quantified at 50 ms after the stimulus. (E) Presynaptic LAR-RPTP deletion has no effect on the coefficient of variation (CV) of AMPAR-EPSCs (left) but cause a modest increase in the CV of NMDAR-EPSCs (right). All data are means ± SEMs. Data comparing two conditions were analyzed by two-tailed unpaired Student’s t-test (for B, ?CRE n = 8/2, CRE n = 7/2, p=0.3711; for C, ?CRE n = 12/3, CRE n = 14/3, p=0.8679 for slope; for D, ?CRE n = 13/3, CRE n = 14/3, *p=0.0246 for NMDA/AMPA ratio; for E, ?CRE n = 13/3, CRE n = 14/3, p=0.3606 for AMPAR-EPSC CV and ?CRE n = 16/4, CRE n = 16/4, *p=0.0439 for NMDAR-EPSC CV). Input-output curves were analyzed by two-way ANOVA for repetitive measurements followed by Bonferroni post-hoc test (for C, ?CRE n = 12/3, CRE n = 14/3, interaction p=0.9833).

Fig 4: Deletion of LAR-RPTPs suppresses synaptic NMDAR- but not AMPAR-mediated responses in cultured hippocampal neurons.All experiments were performed with hippocampal neurons cultured from triple LAR-RPTP cKO mice. Neurons were infected with lentiviruses expressing Cre recombinase (CRE) or a non-functional mutant version of Cre recombinase (ΔCRE, used as control). (A) AMPAR-EPSC amplitudes recorded at −70 mV in presence of PTX are not significantly altered by deletion of all three LAR-RPTPs (left, representative traces; right, summary graphs). (B) NMDAR-EPSC amplitudes recorded at +40 mV in presence of PTX and CNQX are decreased by deletion of all three LAR-RPTPs (left, representative traces; right, summary graphs). (C) Experimental strategy for recording the NMDA/AMPA ratio in hippocampal cultures from triple LAR-RPTP cKO mice infected with ΔCRE (WT) or CRE (KO). (D) NMDAR/AMPAR-EPSC ratios are suppressed by deletion of LAR-RPTPs. AMPAR-EPSCs were recorded at −70 mV in the presence of PTX, and NMDAR-EPSCs were then recorded at +40 mV in the presence of CNQX (left, representative traces; right, summary graphs). (E) Experimental strategy for post-synaptic deletion of LAR-RPTPs in hippocampal cultures. Cultures were sparsely transfected with AAV-synapsin-ΔCRE-EGFP or AAV-synapsin-CRE plasmids at DIV4. Transfected cells expressing EGFP were patched at DIV12-14. (F) NMDA/AMPA ratio in EGFP positive neurons, expressing Cre or ΔCre recombinase, showing that post-synaptic deletion of LAR-RPTPs does not impair AMPAR- or NMDAR-mediated EPSCs. (G) AMPAR-responses elicited by bath-applied AMPA (1 μM) are unchanged by deletion of LAR-RPTPs (left, representative traces; right, summary graphs of peak current densities). (H) NMDAR-responses elicited by bath-applied NMDA (10 μM) and glycine (10 μM) are increased by deletion of LAR-RPTPs (left, representative traces; right, summary graphs of peak current densities). (I–K) Effect of a bath application of 3 μM Ifenprodil, a specific GluN2B blocker, on control hippocampal cultures at different maturation stages (DIV12, blue or DIV15, grey). (I) Schematic of experimental procedures: whole-cell recordings of hippocampal neurons were performed in the presence of CNQX and PTX in the bath to isolate NMDAR-EPSCs that were recorded at +40 mV. Ifenprodil (3 μM) and AP5 were sequentially added to the bath and NMDAR-EPSCs were recorded. (J) Sample traces of NMDAR-EPSCs (% of baseline) as a function of time. Application of Ifenprodil completely blocked NMDAR currents at DIV12 (in blue), but only partially reduced NMDAR currents at DIV15 (in grey), confirming that with maturation neurons switched from GluN2B only containing NMDARs to GluN2A containing NMDARs. Subsequent application of AP5 completely inhibited NMDAR-EPSCs at DIV15; right summary graph depicts the percentage of GluN2A currents at DIV 12 and DIV15. (K) Reduction of NMDAR-EPSCs induced by Ifenprodil was unchanged upon deletion of LAR-RPTPs, suggesting that the NMDAR composition does not depend on LAR-RPTPs. All data are means ± SEMs. Data comparing two conditions were analyzed by two-tailed unpaired Student’s t-test (for A, ΔCRE n = 29/3, CRE n = 27/3, p=0.1993; for B, ΔCRE n = 23/3, CRE n = 22/3, **p=0.0057; for D, ΔCRE n = 18/3, CRE n = 17/3, **p=0.0010; for F, ΔCRE n = 17/3, CRE n = 17/3, p=0.8726; for G, ΔCRE n = 9/2, CRE n = 9/2, p=0.9682; for H, ΔCRE n = 25/3, CRE n = 20/3, *p=0.0212, for K, ΔCRE n = 12/3, CRE n = 15/3, p=0.8891).

Fig 5: Identification of intrinsic neuronal membrane proteins interacting with extracellularly applied Fib-Tau-1N3R Strategy used to purify and identify neuron intrinsic membrane proteins with extracellular domain that interact specifically with Fib-Tau-1N3R. Fib-Tau was labeled 1 h with 10 molar equivalents of NHS-S-S-Biotin. Mouse cortical neuron cultures were exposed for 10 min to biotinylated Fib-Tau (14.4 nM). Fresh protein extracts from those neurons were incubated with streptavidin magnetic beads to pull down Fib-Tau together with their specific protein partners. Unexposed neuron extracts were used as a control. Proteins bound to the streptavidin magnetic beads were eluted with Laemmli buffer and subjected to short migration on a SDS–PAGE gel. After Coomassie blue staining, proteins were subjected to in-gel digestion using trypsin and subsequently identified by nanoLC-MS/MS analysis, using a nanoLC-TripleTOF mass spectrometer. Relative quantification between control and exposed neuron samples was performed using a label-free MS-based approach. Six independent replicates were analyzed.Venn diagram of 968 proteins identified in Fib-Tau pull-downs only (red), in control pull-downs only (gray), or in both samples (overlap). Of the 92 proteins identified in both samples, 45 proteins were significantly enriched in Fib-Tau pull-downs (t-test with P-values < 0.05, Benjamini–Hochberg, fold change > 2).Distribution of the 372 synaptic and membrane protein interactors of Fib-Tau identified in the pull-down experiments. Locations of proteins at the levels of subcellular structures were annotated using the Gene Ontology Cell Component annotation tool of AMIGO 2 (http://amigo.geneontology.org/amigo/landing). Distribution of Fib-Tau interactors in the plasma membrane, pre-synaptic membrane, post-synaptic membrane, pre-synapse, and post-synapse is shown.List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull-downs from neurons exposed to Fib-Tau. For each identified protein, the name of the protein, the gene name, the P-value (t-test with Benjamini–Hochberg correction), and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given. In an independent analysis, after 10-min exposure of neurons to biotinylated Fib-Tau, a cross-linking step was performed during 20 min using 1 mM of DTSSP added in the culture medium, in order to cross-link the protein complexes formed at the cell surface using a membrane impermeable cross-linker. After cross-linking, proteins were analyzed and identified exactly as non-cross-linked samples. Proteins identified with at least two peptides are labeled “+”, and the other are labeled “-”.Co-immunoprecipitation of exogenous biotin-labeled Fib-Tau with a3-NKA, GluA2, and GluN1. a3-NKA, GluA2, and GluN1 were immunoprecipitated using specific antibodies as described in the Materials and Methods section. The presence of Fib-Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin-HRP. A 2.4-, 2.3-, and 1.8-fold enrichment in Tau band intensity is observed in a3-NKA, GluA2, and GluN1 immunoprecipitates, respectively, compared to controls performed with pre-immune goat or rabbit IgGs. Co-immunoprecipitates of exogenous biotin-labeled Fib-Tau with anti-a3-NKA-, GluA2-, and GluN1-specific antibodies were also subjected to SDS–PAGE and Western blot analysis. The presence of Fib-Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin-HRP. Fib-Tau co-immunoprecipitates with a3-NKA and GluA2-AMPA receptor but not with GluN1-NMDA receptor.Network describing the interconnectivity of intrinsic membrane proteins extracellularly exposed (presented in panel D, labeled in yellow) and post-synaptic proteins (proteins with a post-synapse or a post-synaptic membrane annotation, presented in panel C and labeled in blue) that interact with Fib-Tau-1N3R. This Fib-Tau-1N3R interactome was input in the String database (String v10, https://string-db.org/) and exported to Cytoscape (version 3.5.1 at http://www.cytoscape.org/) to visualize interactions between the identified proteins. A total of 121 proteins were evaluated. We set parameters to only detect interactions that were validated experimentally or described in databases. The thickness of the line corresponds to the confidence of interaction (thin lines, > 0.4; medium lines, > 0.7; thick lines, > 0.9).