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The cystine/glutamate antiporter system xc− has been identified as the major source of extracellular glutamate in several brain regions as well as a modulator of neuroinflammation, and genetic deletion of its specific subunit xCT (xCT−/−) is protective in mouse models for age-related neurological disorders. However, the previously observed oxidative shift in the plasma cystine/cysteine ratio of adult xCT−/− mice led to the hypothesis that system xc− deletion would negatively affect life- and healthspan. Still, till now the role of system xc− in physiological aging remains unexplored. We therefore studied the effect of xCT deletion on the aging process of mice, with a particular focus on the immune system, hippocampal function, and cognitive aging. We observed that male xCT−/− mice have an extended lifespan, despite an even more increased plasma cystine/cysteine ratio in aged compared to adult mice. This oxidative shift does not negatively impact the general health status of the mice. On the contrary, the age-related priming of the innate immune system, that manifested as increased LPS-induced cytokine levels and hypothermia in xCT+/+ mice, was attenuated in xCT−/− mice. While this was associated with only a very moderate shift towards a more anti-inflammatory state of the aged hippocampus, we observed changes in the hippocampal metabolome that were associated with a preserved hippocampal function and the retention of hippocampus-dependent memory in male aged xCT−/− mice. Targeting system xc− is thus not only a promising strategy to prevent cognitive decline, but also to promote healthy aging.

Deformation, compression, or stretching of brain tissues cause diffuse axonal injury (DAI) and induce structural and functional alterations of astrocytes, the most abundant cell type in the brain. To gain further insight into the role of mechanically activated astrocytes on neuronal networks, this study was designed to investigate whether cytokines released by mechanically activated astrocytes can affect the growth and synaptic connections of cortical neuronal networks. Astrocytes were cultivated on elastic membranes and subjected to repetitive mechanical insults, whereas well-defined protein micropatterns were used to form standardized neuronal networks. GFAP staining showed that astrocytes were mechanically activated after two cycles of stretch and mesoscale discovery assays indicated that injured astrocytes released four major cytokines. To understand the role of these cytokines, neuronal networks were cultured with the supernatant of healthy or mechanically activated astrocytes, and the individual contribution of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) was studied. We found that the supernatant of two-cycle stretched astrocytes decreased presynaptic terminals and indicated that TNF-α must be considered a key player of the synaptic loss. Furthermore, our results indicate that cytokines released by injured astrocytes significantly modulate the balance between TNFR1 and TNFR2 receptors by enhancing R2 receptors. We demonstrated that TNF-α is not involved in this process, suggesting a predominant role of other secreted cytokines. Together, these results contribute to a better understanding of the consequences of repetitive astrocyte deformations and highlight the role of inflammatory signaling pathways in synaptic plasticity and modulation of TNFR1 and TNFR2 receptors.

The astrocytic cystine/glutamate antiporter system xc− represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc−, xCT (xCT−/− mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT−/− mice. A proteomic and kinomic screen of the striatum of xCT−/− mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT−/− mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc− plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.

Worldwide, approximately 27 million people are affected by Alzheimer’s disease (AD). AD pathophysiology is believed to be caused by the deposition of the β-amyloid peptide (Aβ). Aβ can reduce long-term potentiation (LTP), a form of synaptic plasticity that is closely associated with learning and memory and involves postsynaptic glutamate receptor phosphorylation and trafficking. Moreover, Aβ seems to be able to reduce glutamatergic transmission by increasing the endocytosis of NMDA receptors. Trace amines (TAs) are biogenic amines that are structurally similar to monoamine neurotransmitters. TAs bind to G protein-coupled receptors, called TAARs (trace amine-associated receptors); the best-studied member of this family, TAAR1, is distributed in the cortical and limbic structures of the CNS. It has been shown that the activation of TAAR1 can rescue glutamatergic hypofunction and that TAAR1 can modulate glutamate NMDA receptor-related functions in the frontal cortex. Several lines of evidence also suggest the pro-cognitive action of TAAR1 agonists in various behavioural experimental protocols. Thus, we studied, in vitro, the role of the TAAR1 agonist RO5256390 on basal cortical glutamatergic transmission and tested its effect on Aβ-induced dysfunction. Furthermore, we investigated, in vivo, the role of TAAR1 in cognitive dysfunction induced by Aβ infusion in Aβ-treated mice. In vitro data showed that Aβ 1–42 significantly decreased NMDA cell surface expression while the TAAR1 agonist RO5256390 promoted their membrane insertion in cortical cells. In vivo, RO5256390 showed a mild pro-cognitive effect, as demonstrated by the better performance in the Y maze test in mice treated with Aβ. Further studies are needed to better understand the interplay between TAAR1/Aβ and glutamatergic signalling, in order to evaluate the eventual beneficial effect in different experimental paradigms and animal models. Taken together, our data indicate that TAAR1 agonism may provide a novel therapeutic approach in the treatments of disorders involving Aβ-induced cognitive impairments, such as AD.

The cystine/glutamate antiporter system x

Traumatic brain injury (TBI) remains one of the leading causes of mortality and morbidity worldwide. Despite its high prevalence and extensive efforts to develop neuroprotective therapies, effective treatments for TBI are still limited. Among important neuronal damages, TBI induces structural and functional alterations of astrocytes, the most abundant cell type in the brain. Injured astrocytes respond in diverse mechanisms that result in reactive astrogliosis and are involved in the physiopathological mechanisms of TBI in an extensive and sophisticated manner. The establishment of effective neuroprotective treatments for TBI requires to better understand the complex biochemical interactions between activated astrocytes and neurons that contribute to the secondary injury. To address this challenge, we studied in vitro the role of mechanically injured astrocytes on the growth and synaptic connections of cortical neuronal networks of controlled architectures grown on well-defined protein micropatterns. Astrocytes were cultivated on elastic membranes and mechanically activated by stretching cycles. The culture media of healthy or activated astrocytes was then introduced on neuronal networks. We analyzed the neuronal viability, the neurite growth and the synaptic density of neuronal networks to understand the role of the inflammatory molecules secreted by mechanically activated astrocytes. Furthermore, we cultivated neuronal networks during 13 days with different doses of TNF-α in order to decipher its individual contribution among the other cytokines. Here we show that the ratio of tubulin to synapsin area was significantly higher in neuronal networks treated with either 4 or 2 doses of TNF-α, suggesting that TNF-α can promote the tubulin polymerization process. Assuming that TNF-α can bind to either TNFR1 or TNFR2 receptors, which lead respectively to the cell survival or the cell apoptosis, we studied the modulation of the both TNF-α receptors in response to the medium of mechanically activated astrocytes and different doses of TNF-α. Our findings indicate that the amount of both receptors increases with the maturation of the network. In addition, we observed a significant modulation of the amount of TNFR1 and TNFR2 in response to the media of injured astrocytes that leads to a large imbalance between both receptors, suggesting an important role for TNFα-signaling in the physiopathological mechanisms of TBI.

The cystine/glutamate antiporter system xc-, with xCT as specific subunit, exchanges intracellular glutamate for extracellular cystine. It is highly expressed in the central nervous system, mainly on astrocytes, and has been shown to be the major source of extracellular glutamate in various regions of the brain, such as the striatum and hippocampus. This extrasynaptically released glutamate can affect synaptic neurotransmission. As the physiological function of system xc- in the central nervous system remains poorly understood, we studied how system xc- regulates transmission at corticostriatal synapses, one of the two major types of striatal excitatory synapses. Electrophysiological recordings identified a significant decrease in the amplitude of striatal field excitatory postsynaptic potentials in mice genetically lacking xCT (xCT-/- mice) following stimulation of corticostriatal fibers. Further, using electron microscopy, we observed depletion of glutamate immunogold labeling from corticostriatal terminalsand their corresponding dendritic spines in xCT-/- mice. Genetic deletion of xCT did not, however, affect the morphology of corticostriatal synapses, the density of cortical innervation or the density of dendritic spines in the striatum. Proteomic analysis revealed decreased expression of a wide range of proteins involved in regulating presynaptic neurotransmitter release in the striatum of xCT-/- mice, including synaptophysin, VGLUT1, and members of the synapsin, septin, and syntaxin families. In addition, kinome profiling identified changes in striatal serine/threonine kinase activity, highlighting ERK signaling as a possible node of kinase dysregulation in xCT-/- mice. Finally, we evaluated the effect of the disturbed corticostriatal communication on the behavioral phenotype of the xCT-/- mice. In the marble burying test, a paradigm sensitive to changes in corticostriatal function, we measured a significant increase in repetitive digging behavior in xCT-/- mice. Whereas spontaneous grooming behavior was not affected by genetic deletion of xCT, we have preliminary findings from the reciprocal interaction and three-chamber test, suggesting aberrant social behavior. Together, our results identify system xc- as a modulator of corticostriatal synaptic transmission that may be relevant to neuropsychiatric disorders characterized by corticostriatal dysfunction and repetitive behavior, such as obsessive-compulsive behavior and autism.

System xc-, with xCT as specific subunit, is an astrocytic antiporter that imports cystine in exchange for glutamate. System xc- is enhanced following oxidative stress and inflammation1, both present in the diseased or aged brain2. This upregulation might induce or further drive neurological dysfunction, as it could decrease the threshold for glutamate toxicity (by releasing glutamate)3 and modulate neuroinflammation (by driving the pro-inflammatory microglial phenotype)4, 5. Although inhibition of system xc- has been proposed as a therapeutic strategy for diverse neurological disorders (some age-related), reports on its function in physiological aging are sparse. We first studied the effect of genetic xCT deletion (xCT-/- mice) on life- and health-span. Next, we investigated the effect of aging on hippocampal xCT expression as well as the effect of xCT deletion on hippocampal aging (3-4 month versus 18-19-month old mice). As glutamate released via system xc- into the extrasynaptic space, could modulate synaptic transmission, we hypothesized that absence of system xc- might affect age-induced hippocampal deficits. We therefore compared age-related changes in hippocampal neurotransmission (slice electrophysiology), long-term potentiation (LTP) as well as hippocampus-dependent memory (Barnes maze, novel object location recognition task (NOLR)) between xCT-/- mice and their xCT+/+ littermates. xCT-/- mice have an increased life-span without deterioration of health-span, compared to xCT+/+ mice. Next, although no age-induced changes in xCT protein expression were seen in the brain of xCT+/+ mice (C57BL/6J background; 3-4 month versus 20-24-month old mice), xCT mRNA was significantly increased in hippocampus of 13-month old compared to 9-month old SAMP8 mice (model for accelerated aging). Aging prolongs the learning phase in the Barnes maze task for both genotypes. However, contrary to aged xCT+/+ mice and comparable to adult mice, the majority of aged xCT-/- mice use the hippocampus-dependent direct search strategy in the Barnes maze set-up at the end of the 5-day training session and they preserve this memory till 5 days after the last training session. In the NOLR, loss of system xc- induces impairment of spatial memory in adult mice. However, whereas aging negatively affects performance in the NOLR task in xCT+/+ mice, this was not the case for xCT-/- littermates and aged xCT-/- mice even perform better compared to adult xCT-/- mice. These data suggest that the hippocampal aging process is fundamentally different in mice lacking system xc-. In line with our behavioral data, basal hippocampal neurotransmission is indeed reduced in adult xCT-/- mice, while the age-related decrease in basal synaptic transmission as well as the age-induced changes in LTP that are observed in xCT+/+ mice, are prevented in the absence of system xc-. To conclude, our results demonstrate that absence of system xc- increases life-span and confers protection against age-related hippocampal dysfunction. Indeed, while system xc- seems to be important for hippocampal function in adult animals, it can become harmful during the aging process, thereby contributing to age-related memory decline.

Long-term potentiation (LTP) remains the most widely accepted model for learning and memory. In accordance with this belief, the temporal differentiation of LTP into early and late phases is accepted as reflecting the differentiation of short-term and long-term memory. Moreover, during the past 30 years, protein synthesis inhibitors have been used to separate the early, protein synthesis-independent (E-LTP) phase and the late, protein synthesis-dependent (L-LTP) phase. However, the role of these proteins has not been formally identified. Additionally, several reports failed to show an effect of protein synthesis inhibitors on LTP. In this review, a detailed analysis of extensive behavioral and electrophysiological data reveals that the presumed correspondence of LTP temporal phases to memory phases is neither experimentally nor theoretically consistent. Moreover, an overview of the time courses of E-LTP in hippocampal slices reveals a wide variability ranging from de novosynthesis of plasticity-related proteins. This availability is determined by protein turnover kinetics, which is regulated by previous and ongoing electrical activities and by energy store availability.

System xc- is a plasma membrane amino acid antiporter, of mainly glial origin, that couples the import of cystine with the export of glutamate. System xc- (specific subunit xCT) contributes substantially to ambient extracellular glutamate levels in various regions of the brain, including the striatum and hippocampus. Despite the fact that system xc- is highly expressed in the brain and is a proposed therapeutic target for various neurological disorders, including Parkinson’s disease, Alzheimer’s disease, multiple sclerosis and epilepsy, its function under physiological conditions in the central nervous system remains poorly understood. By acting as a source of glial extrasynaptic glutamate, system xc- might modulate synaptic transmission as a mechanism of neuro-glial communication. Previous electrophysiological findings indicate that system xc- delivered glutamate can inhibit excitatory synaptic neurotransmission in the cortico-accumbens pathway (Moran et al. J Neurosci. 2005; 25:6389-93) and at hippocampal CA3-CA1 synapses (Williams et al. J Neurosci. 2014; 34:16093-102). In order to gain further insight into the proposed function of system xc- as modulator of synaptic transmission, we carried out single section electron microscopy analyses of cortico-striatal excitatory synapses in adult xCT knockout (xCT-/-) and xCT wildtype (xCT+/+) mice. Our preliminary findings reveal depletion of glutamate immunogold labeling from presynaptic terminals of xCT-/- mice, an increase in the head diameter and area of spines contacted by asymmetric synapses, enlargement of the postsynaptic density and an increased occurrence of spinules, post-synaptic elements of high efficacy. These synaptic changes occurred in the absence of differences in the total density of cortico-striatal synapses or dendritic spines in the dorsolateral striatum of xCT-/- vs. xCT+/+ mice. Our results suggest the involvement of both presynaptic and postsynaptic forms of synaptic strength regulation via system xc-. Whether the structural changes we observedindicate a global increase in cortico-striatal transmission in xCT-/- mice, or conversely, represent forms of synaptic compensation, is being addressed by electrophysiological measures of excitatory post-synaptic currents in xCT-/- mice after cortical stimulation. Together, our findings shed new light on the re-organization of the glutamatergic system after genetic deletion of system xc-, and confirm the involvement of this antiporter in the control of synaptic strength in vivo.

In vitro and in vivo studies suggest that the astrocytic adrenergic signalling enhances glycogenolysis which provides energy to be transported to nearby cells and in the form of lactate. This energy source is important for motor and cognitive functioning. While it is suspected that the beta 2-adrenergic receptor on astrocytes might contribute to this energy balance, it has not yet been shown conclusively in vivo. Inducible astrocyte specific beta 2-adrenergic receptor knock-out mice were generated by crossing homozygous beta 2-adrenergic receptor floxed mice (Adrb2(flox)) and mice with heterozygous tamoxifen-inducible Cre recombinase-expression driven by the astrocyte specific L-glutamate/L-aspartate transporter promoter (GLAST-CreERT2). Assessments using the modified SHIRPA (SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment) test battery, swimming ability test, and accelerating rotarod test, performed at 1, 2 and 4 weeks, 6 and 12 months after tamoxifen (or vehicle) administration did not reveal any differences in physical health or motor functions between the knock-out mice and controls. However deficits were found in the cognitive ability of aged, but not young adult mice, reflected in impaired learning in the Morris Water Maze. Similarly, long-term potentiation (LTP) was impaired in hippocampal brain slices of aged knock-out mice maintained in low glucose media. Using microdialysis in cerebellar white matter we found no significant differences in extracellular lactate or glucose between the young adult knock-out mice and controls, although trends were detected. Our results suggest that beta 2-adrenergic receptor expression on astrocytes in mice may be important for maintaining cognitive health at advanced age, but is dispensable for motor function.

Background System xc- is a plasma membrane amino acid antiporter, of mainly glial origin, that couples the import of cystine with the export of glutamate. System xc- (specific subunit xCT) contributes substantially to ambient extracellular glutamate levels in various regions of the brain, including the striatum and hippocampus. Despite the fact that system xc- is highly expressed in the brain and is a proposed therapeutic target for various neurological disorders, its function under physiological conditions in the central nervous system remains poorly understood. By acting as a source of glial extrasynaptic glutamate, system xc- might modulate synaptic transmission as a mechanism of neuro-glial communication. Previous electrophysiological findings indicate that system xc- delivered glutamate can inhibit excitatory synaptic neurotransmission in the corticoaccumbens pathway and at hippocampal CA3-CA1 synapses. To gain further insight into the proposed function of system xc- as modulator of synaptic transmission, we here focus on corticostriatal synapses. Methods Single section electron microscopy was carried out on VGLUT1-pre-embed and glutamate immunogold post-embed labeled slices of the dorsolateral striatum of xCT+/+ and xCT-/- mice. Various parameters related to the pre- and post-synaptic compartments were integrated on the obtained electron micrographs, including glutamate immunogold density in the presynaptic terminal and spine, area of the terminal and spine, measures of the postsynaptic density (PSD) (length, area, thickness, and maximum thickness), percentage of PSDs showing perforations, and width of the synaptic cleft. Electrophysiological measures of corticostriatal transmission were obtained by recording the amplitude of field excitatory postsynaptic potentials (fEPSPs) after stimulation of corticostriatal fibers. Finally, grooming behavior was compared between xCT-/- and xCT+/+ littermates. Results Genetic deletion of xCT led to depletion of glutamate immunogold labeling from corticostriatal terminals and their corresponding dendritic spines. Absence of xCT did not, however, affect the morphology of corticostriatal synapses, as evaluated by the area of the terminals and spines, size of the PSD, and width of the synaptic cleft. Similarly, no changes could be observed in the density of VGLUT1-positive synapses, indicating normal cortical innervation and spine density. Electrophysiological recordings revealed decreased amplitude of fEPSPs in xCT-/- mice after stimulation of corticostriatal fibers. Preliminary investigations revealed that this reduced response can be rescued by restoring physiological levels of glutamate to xCT-/- slices. Changes in corticostriatal transmission were not reflected in aberrant grooming behavior in xCT-/- mice; we could not observe any difference in the total grooming duration, the number of grooming bouts, the average bout duration or the latency to onset to grooming between xCT-/- and xCT+/+ mice. Discussion Contrary to available evidence at hippocampal and corticoaccumbens pathways, our findings indicate a positive effect of system xc- on basal synaptic transmission at corticostriatal synapses. The decreased response we observed after stimulation of corticostriatal fibers in xCT-/- mice was accompanied by depletion of glutamate immunogold labeling from corticostriatal terminals, suggesting a possible defect in presynaptic glutamate handling. Given the strong decrease (70%) in extracellular glutamate levels previously reported in this strain of mice, we hypothesize that the decreased presynaptic glutamate labeling in xCT-/- mice is related to a loss of extracellular glutamate needed to supply terminals for proper excitatory transmission. This hypothesis is supported by our preliminary results showing increased responses in xCT-/- slices after restoring physiological levels of glutamate. Together, our findings shed new light on the role of system xc- in controlling synaptic transmission, and suggest that it may play an important role in supplying presynaptic terminals with glutamate as an alternative mechanism to the glutamate-glutamine cycle. As a novel modulator of corticostriatal transmission, system xc- may be of interest as a possible therapeutic target for disorders with a corticostriatal component, such as schizophrenia or obsessive-compulsive disorder.

In the CA1 region of mouse hippocampal slices, a strong tetanic stimulation triggers a long-lasting long-term potentiation (L-LTP), which requires transcription for the development of its late phase. Nevertheless, we were able to elicit such an L-LTP in CA1 dendrites separated from their somas provided that we restricted our investigations to isolated dendrites where a very robust early LTP was triggered. This particular type of L-LTP, which relied on translation of preexisting messenger RNAs - as it was blocked by anisomycin - could not be captured by another pathway activated only by a weak tetanic stimulation. This suggests that the plasticity-related proteins resulting from translation of messenger RNAs in dendrites cannot pass from the synaptic site where they were synthesized to another one.

α-calcium/calmodulin-dependent protein kinase (αCaMKII) T286-autophosphorylation provides a short-term molecular memory that was thought to be required for LTP and for learning and memory. However, it has been shown that learning can occur in αCaMKII-T286A mutant mice after a massed training protocol. This raises the question of whether there might be a form of LTP in these mice that can occur without T286 autophosphorylation. In this study, we confirmed that in CA1 pyramidal cells, LTP induced in acute hippocampal slices, after a recovery period in an interface chamber, is strictly dependent on postsynaptic αCaMKII autophosphorylation. However, we demonstrated that αCaMKII-autophosphorylation-independent plasticity can occur in the hippocampus but at the expense of synaptic specificity. This nonspecific LTP was observed in mutant and wild-type mice after a recovery period in a submersion chamber and was independent of NMDA receptors. Moreover, when slices prepared from mutant mice were preincubated during 2 h with rapamycin, high-frequency trains induced a synapse-specific LTP which was added to the nonspecific LTP. This specific LTP was related to an increase in the duration and the amplitude of NMDA receptor-mediated response induced by rapamycin.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection. The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

Current thinking about LTP triggered in the area CA1 of hippocampal slices is ruled by two “dogmas”: (1) A single train of high-frequency stimulation is sufficient to trigger short-lasting LTP (1 – 3 h), whereas multiple trains are required to induce long-lasting LTP (L-LTP, more than 4 h). (2) The development of the late phase of L-LTP requires the synthesis of new proteins. In this study, we found that a single high-frequency train could trigger an LTP lasting more than 8 h that was not affected by either anisomycin or cycloheximide (two inhibitors of protein synthesis). We ascertained that the induction of this L-LTP made use of the same mechanisms as those usually reported to be involved in LTP induction: it was dependent on NMDA receptors and on the activation of two “core” kinases, CaMKII and PI3K. These findings call into question the two “dogmas” about LTP.

Long-term memory (LTM) formation has been linked with functional strengthening of existing synapses and other processes including de novo synaptogenesis. However, it is unclear whether synaptogenesis can contribute to LTM formation. Here, using α-calcium/calmodulin kinase II autophosphorylation-deficient (T286A) mutants, we demonstrate that when functional strengthening is severely impaired, contextual LTM formation is linked with training-induced PSD95 up-regulation followed by persistent generation of multiinnervated spines, a type of synapse that is characterized by several presynaptic terminals contacting the same postsynaptic spine. Both PSD95 up-regulation and contextual LTM formation in T286A mutants required signaling by the mammalian target of rapamycin (mTOR). Furthermore, we show that contextual LTM resists destabilization in T286A mutants, indicating that LTM is less flexible when synaptic strengthening is impaired. Taken together, we suggest that activation of mTOR signaling, followed by overexpression of PSD95 protein and synaptogenesis, contributes to formation of invariant LTM when functional strengthening is impaired.

Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected 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, 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, including in TauP301 mutants. Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected 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, 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, including in TauP301 mutants. This work was supported by the Belgian Fonds National pour la Recherche Scientifique—Fonds de la Recherche Scientifique (FNRS-FRS; Qualified Researcher, Impulse Financing, Research Credits), by Interuniversity Attraction Poles ProgrammeBelgian State-Belgian Science Policy, The Belgian Fonds de la Recherche Scientifique Médicale, by the Institute for the Promotion of Innovation by Science and Technology (IWT) in Flanders (IWT O&O), Belgium.

The ability to construct easily in vitro networks of primary neurons organized with imposed topologies is required for neural tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. However, accumulating evidence suggests that the mechanical properties of the culture matrix can modulate important neuronal functions such as growth, extension, branching and activity. Here we designed robust and reproducible laminin-polylysine grid micropatterns on cell culture substrates that have similar biochemical properties but a 100-fold difference in Young's modulus to investigate the role of the matrix rigidity on the formation and activity of cortical neuronal networks. We found that cell bodies of primary cortical neurons gradually accumulate in circular islands, whereas axonal extensions spread on linear tracks to connect circular islands. Our findings indicate that migration of cortical neurons is enhanced on soft substrates, leading to a faster formation of neuronal networks. Furthermore, the pre-synaptic density was two times higher on stiff substrates and consistently the number of action potentials and miniature synaptic currents was enhanced on stiff substrates. Taken together, our results provide compelling evidence to indicate that matrix stiffness is a key parameter to modulate the growth dynamics, synaptic density and electrophysiological activity of cortical neuronal networks, thus providing useful information on scaffold design for neural tissue engineering. (C) 2016 Elsevier Ltd. All rights reserved.