Sagittal sections (40?m) were lower on the vibrating HM650V microtome (Thermo Fisher Scientific, Waltham, MA, USA). neurons. Electrophysiological evaluation in organotypic hippocampal pieces of Tau transgenic mice proven impaired synaptic transmitting and impaired long-term potentiation pursuing Tau-seed induced Tau-aggregation. Intracerebral shot of Tau-seeds in TauP301S mice, triggered prion-like growing of Tau-pathology through linked neuroanatomical pathways. Electrophysiological analysis exposed impaired synaptic plasticity in hippocampal CA1 area 6?weeks after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, assessed in the thing recognition check 6?weeks post-seeding. On the other hand, Tau-seeding in basal ganglia and following growing through linked neuronal systems involved with engine control functionally, led to motoric deficits shown in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, Benzocaine ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, Benzocaine including in TauP301 mutants. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1413-4) contains supplementary material, which is available to authorized users. Tau-PFFs (synthetic preformed fibrils) or Tau-seeds were generated as described [26, 35]. Briefly, truncated human Tau fragments (WT-Tau/P301L-Tau) containing the four repeat domain [K18; Q244-E372 (4RTau)], N-terminally Myc-tagged were produced in PFFs induced Tau-aggregation in vitro was essentially performed as previously . Sonicated Tau-seeds (10?M) were added to Bio-PORTER single use tubes (AMS Biotechnology, Milton, UK) and added to optiMEM-washed transiently transfected HEK293 (QBI) cells, 24?h post-transfection with Tau (P301L or WT). Primary cortical neuronal cultures (PNC) were generated as described previously , from P0 heterozygous TauP301S pups or non-transgenic (WT) littermates. Tau-seeds (10?nM) were added at DIV3 and DIV6, and primary neurons were used for calcium imaging at DIV13 and subsequently extracted or fixed for further analysis. Organotypic hippocampal slices (OC) were generated using previously described protocols [21, 64]. Briefly, hippocampal slices were generated at P6 from heterozygous TauP301S mice and non-transgenic littermates. Tau-seeds (1?L; 333?M) were gently added on top of hippocampal slices at DIV3 and DIV6, and slices were analyzed electrophysiologically, biochemically and immunohistologically 10?days after seeding. Sonicated pre-aggregated Tau-PFFs (5?L; 333?M) or vehicle without seeds (5?L) were injected in 3?months old mice. Stereotactic injections Benzocaine were performed in the hippocampal CA1 region (A/P, ?2.0; L, +1.4; D/V, ?1.2), frontal cortex (A/P, +2.0; L, +1.4; D/V, ?1.0), in entorhinal cortex (A/P, ?4.8; L, ?3.0; D/V, ?3.7) and substantia nigra (A/P, ?4.8; P/A, angle 16; L, ?1.1; D/V, ?4.7) millimeter relative to bregma , using a 10?L Hamilton syringe at a speed of 1 1?L per min. Biochemical analysis For immunoblotting analysis brain regions were dissected and snap-frozen in liquid nitrogen, and subsequently differential extraction of total homogenates, sarkosyl soluble and sarkosyl insoluble fractions was performed as previously described [67, 68] and in supplemental data. Similar extraction procedures were used for primary neurons and organotypic cultures as described in supplemental data. Proteins were quantified using BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). For dot blotting equal amounts of total homogenates were spotted on a nitrocellulose membrane and subsequently immunoblotted using T22 [ABN454 Anti-Tau (T22) oligomeric CENPA antibody; EMD Millipore], against oligomeric Tau . Analysis under non-denaturating and non-reducing conditions was performed on 4-16?% Bis-Tris Native Benzocaine Page. For immunoblotting equal amounts of proteins were loaded.