The various effect may be because of additional actions of high Mg2+, such as for example inhibition of transmitter release [23], which can affect spine thickness. receptor function) in conjunction with a control siRNA still exhibited late-LTP, while siRNA to Stau1 was effective in blocking late-LTP still. Our outcomes indicate that Stau1 participation in backbone morphogenesis would depend on ongoing NMDA receptor-mediated plasticity, but its effects on late-LTP are independent of the noticeable changes. These results clarify the function of Stau1-reliant mRNA legislation in physiological and morphological adjustments root long-term synaptic plasticity in pyramidal cells. solid course=”kwd-title” Keywords: Schaffer guarantee synapses, RNA transportation, later LTP, spontaneous activity-driven potentiation, backbone morphogenesis Launch Localization of mRNAs to synaptic sites and their following translation have surfaced as important systems adding to synapse-specific plasticity [1,2]. Hence, mRNA binding protein (RBPs), which are fundamental players in the transportation of mRNAs, could be selectively implicated in a variety of types of plasticity that rely on the transportation and regional translation of particular transcripts. Staufen (Stau) [3,4], delicate mental retardation proteins (FMRP) [5,6], zipcode-binding protein [7] and cytoplasmic polyadenyation component binding proteins (CPEB) [8,9] are RBPs regarded as implicated in mRNA dendritic translation and localization in neurons. Notably, Stau is certainly implicated in legislation of mRNAs necessary for storage development in Aplysia and Drosophila [10,11]. In mammals, both members from the Stau family members, Stau2 and Stau1, can be found in distinctive ribonucleoprotein (RNP) complexes [12] and associate with different mRNAs [13]. Stau1 is necessary for the transportation of mRNAs essential for long-term potentiation at hippocampal synapses, as knockdown of Stau1 impaired dendritic transportation of CaMKII mRNA in hippocampal neurons [3]. Furthermore, downregulation of Stau1 also avoided the translation-dependent past due stage LTP (late-LTP) induced by forskolin in CA1 pyramidal cells. Nevertheless, the translation-independent early stage LTP was intact, recommending an essential function of Stau1-reliant mRNA legislation in proteins synthesis connected with late-LTP [14]. Oddly enough, we lately discovered that Stau2-reliant legislation of mRNA was needed for translation-dependent mGluR long-term despair particularly, uncovering selective systems of mRNA legislation for different types of translation-dependent long-term synaptic plasticity [15]. Long-term adjustments in synaptic function are connected with adjustments in dendritic spines [16,17]. Certainly, we discovered that, in colaboration with the impairment in late-LTP, Stau1 knockdown led to a change from regular brief spines to much longer thin spines, recommending a job in the development and/or maintenance of older spine form [14]. Nevertheless, since a form of NMDA-mediated plasticity, referred to as spontaneous activity-driven potentiation (SAP) [18], may be ongoing in our slice culture conditions and induce changes in spine shape [19-21], it is unknown whether the effects of Stau1 knockdown on late-LTP were due to its actions on spine morphogenesis, or vice versa. Thus, our aims were to test directly if preventing SAP by blocking NMDAR function (or elevating extracellular Mg2+) would influence the changes in dendritic spine morphology induced by Stau1 knockdown, and whether the changes induced by blocking SAP were in turn required for the effect on Stau1 knockdown on late-LTP. We found that while Stau1 is usually involved in spine morphogenesis through NMDAR-mediated SAP, the change in spine morphogenesis was not important for the effect of Stau1 on late-LTP. PQR309 Methods Organotypic hippocampal slice cultures All experiments were done in accordance with animal care guidelines at Universit de Montral, with the approval of the ethics committee at Universit de Montral (CDEA #10-003), and followed internationally recognized guidelines. Organotypic hippocampal slices were prepared and maintained in culture as previously described [14,22]. siRNAs and transfections siRNA target sequences for rat were as described [14]. Biolistic transfection of neurons in organotypic slice cultures was performed using a Helios gene gun (Bio-Rad, CA) following manufacturer’s instructions as previously.In the context of late-LTP, the mRNAs that are translated can consist of previously transcribed plasticity-related mRNAs that were transported constitutively to synapses prior to LTP induction [27] or newly transcribed mRNAs that need to be transported to the activated synapse for local translation [30]. Stau1 knockdown in the presence of NMDA receptor blockade (or high Mg2+) has no further effect on spine shape and size. Moreover, the effect of Stau1 knockdown on late-LTP cannot be explained by these effects, since when tested in normal medium, slice cultures that had been treated with high Mg2+ (to impair NMDA receptor function) in combination with a control siRNA still exhibited late-LTP, while siRNA to Stau1 was still effective in blocking late-LTP. Our results indicate that Stau1 involvement in spine morphogenesis is dependent on ongoing NMDA receptor-mediated plasticity, but its effects on late-LTP are impartial of these changes. These findings clarify the role of Stau1-dependent mRNA regulation in physiological and morphological changes underlying long-term synaptic plasticity in pyramidal cells. strong class=”kwd-title” Keywords: Schaffer collateral synapses, RNA transport, late LTP, spontaneous activity-driven potentiation, spine morphogenesis Introduction Localization of mRNAs to synaptic sites and their subsequent translation have emerged as important mechanisms contributing to synapse-specific plasticity [1,2]. Thus, mRNA binding proteins (RBPs), which are key players in the transport of mRNAs, may be selectively implicated in various forms of plasticity that depend on the transport and local translation of specific transcripts. Staufen (Stau) [3,4], fragile mental retardation protein (FMRP) [5,6], zipcode-binding proteins [7] and cytoplasmic polyadenyation element binding protein (CPEB) [8,9] are RBPs known to be implicated in mRNA dendritic localization and translation in neurons. Notably, Stau is usually implicated in regulation of mRNAs required for memory formation in Drosophila and Aplysia [10,11]. In mammals, the two members of the Stau family, Stau1 and Stau2, are present in distinct ribonucleoprotein (RNP) complexes [12] and associate with different mRNAs [13]. Stau1 is required for the transport of mRNAs necessary for long-term potentiation at hippocampal synapses, as knockdown of Stau1 impaired dendritic transport of CaMKII mRNA in hippocampal neurons [3]. Moreover, downregulation of Stau1 also prevented the translation-dependent late phase LTP (late-LTP) induced by forskolin in CA1 pyramidal cells. However, the translation-independent early phase LTP was intact, suggesting an essential role of Stau1-dependent mRNA regulation in protein synthesis associated with late-LTP [14]. Interestingly, we recently found that Stau2-dependent regulation of mRNA was essential specifically for translation-dependent mGluR long-term depression, uncovering selective mechanisms of mRNA regulation for different forms of translation-dependent long-term synaptic plasticity [15]. Long-term changes in synaptic function are associated with changes in dendritic spines [16,17]. Indeed, we found that, in association with the impairment in late-LTP, Stau1 knockdown resulted in a shift from regular short spines to longer thin spines, suggesting a role in the formation and/or maintenance of mature spine shape [14]. However, since a form of NMDA-mediated plasticity, referred to as spontaneous activity-driven potentiation (SAP) [18], may be ongoing in our slice culture conditions and induce changes in spine shape [19-21], it is unknown whether the effects of Stau1 knockdown on late-LTP were due to its actions on spine morphogenesis, or vice versa. Thus, our aims were to test directly if preventing SAP by blocking NMDAR function (or elevating extracellular Mg2+) would influence the changes in dendritic spine morphology induced by Stau1 knockdown, and whether the changes induced by blocking SAP were in turn required for the effect on Stau1 knockdown on late-LTP. We found that while Stau1 is involved in spine morphogenesis through NMDAR-mediated SAP, the change in spine morphogenesis was not important for the effect of Stau1 on late-LTP. Methods Organotypic hippocampal slice cultures All experiments were done in accordance with animal care guidelines at Universit de Montral, with the approval of the ethics committee at Universit de Montral (CDEA #10-003), and followed internationally recognized guidelines. Organotypic hippocampal slices were prepared and maintained in culture as previously described [14,22]. siRNAs and transfections siRNA target sequences for rat were as described [14]. Biolistic transfection of neurons in organotypic slice cultures was performed using a Helios gene.Once again, there was no further effect of high Mg2+ or AP5 medium in siRNA-STAU1 transfected cells (relative to siRNA-CTL in high Mg2+ or AP5, respectively). late-LTP, while siRNA to Stau1 was still effective in blocking late-LTP. Our results indicate that Stau1 involvement in spine morphogenesis is dependent on ongoing NMDA receptor-mediated plasticity, but its effects on late-LTP are independent of these changes. These findings clarify the role of Stau1-dependent mRNA regulation in physiological and morphological changes underlying long-term synaptic plasticity in pyramidal cells. strong class=”kwd-title” Keywords: Schaffer collateral synapses, RNA transport, late LTP, spontaneous activity-driven potentiation, spine morphogenesis Introduction Localization of mRNAs to synaptic sites and their subsequent translation have emerged as important mechanisms contributing to synapse-specific plasticity [1,2]. Thus, mRNA binding proteins (RBPs), which are key players in the transport of mRNAs, may be selectively implicated in various forms of plasticity that depend on the transport and local translation of specific transcripts. Staufen (Stau) [3,4], fragile mental retardation protein (FMRP) [5,6], zipcode-binding proteins [7] and cytoplasmic polyadenyation element binding protein (CPEB) [8,9] are RBPs known to be implicated in mRNA dendritic localization and translation in neurons. Notably, Stau is implicated in regulation of mRNAs required for memory formation in Drosophila and Aplysia [10,11]. In mammals, the two members of the Stau family, Stau1 and Stau2, are present in distinct ribonucleoprotein (RNP) complexes [12] and associate with different mRNAs [13]. Stau1 is required for the transport of mRNAs necessary for long-term potentiation at hippocampal synapses, as knockdown of Stau1 impaired dendritic transport of CaMKII mRNA in hippocampal neurons [3]. Moreover, downregulation of Stau1 also prevented the translation-dependent late phase LTP (late-LTP) induced by forskolin in CA1 pyramidal cells. However, the translation-independent early phase LTP was intact, suggesting an essential role of Stau1-dependent mRNA regulation in protein synthesis associated with late-LTP [14]. Interestingly, we recently found that Stau2-dependent regulation of mRNA was essential specifically for translation-dependent mGluR long-term depression, uncovering selective mechanisms of mRNA regulation for different forms of translation-dependent long-term synaptic plasticity [15]. Long-term changes in synaptic function are associated with changes in dendritic spines [16,17]. Indeed, we found that, in association with the impairment in late-LTP, Stau1 knockdown resulted in a shift from regular short spines to longer thin spines, suggesting a role in the formation and/or maintenance of adult spine shape [14]. However, since a form of NMDA-mediated plasticity, referred to as spontaneous activity-driven potentiation (SAP) [18], may be ongoing in our slice culture conditions and induce changes in spine shape [19-21], it is unknown whether the effects of Stau1 knockdown on late-LTP were due to its actions on spine morphogenesis, or vice versa. Therefore, our aims were to test directly if avoiding SAP by obstructing NMDAR function (or elevating extracellular Mg2+) would influence the changes in dendritic spine morphology induced by Stau1 knockdown, and whether the changes induced by obstructing SAP were in turn required for the effect on Stau1 knockdown on late-LTP. We found that while Stau1 is definitely involved in spine morphogenesis through NMDAR-mediated SAP, the switch in spine morphogenesis was not important for the effect of Stau1 on late-LTP. Methods Organotypic hippocampal slice cultures All experiments were done in accordance with animal care recommendations at Universit de Montral, with the approval of the ethics committee at Universit de Montral (CDEA #10-003), and adopted internationally recognized recommendations. Organotypic hippocampal slices were prepared and managed in tradition as previously explained [14,22]. siRNAs and transfections siRNA target sequences for rat were as explained [14]. Biolistic transfection of neurons in organotypic slice ethnicities was performed using a Helios gene gun (Bio-Rad, CA) following manufacturer’s instructions as previously [14,22]. Electrophysiological recordings and cell imaging experiments were performed 48 hours after transfection and the experimenter was blind to transfection treatments. Electrophysiology Individual slice cultures were transferred to a submerged-type recording chamber continually perfused (at 1-2 ml/min) with artificial cerebrospinal fluid (ACSF) composed of (in mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.3 MgSO4, 26 NaHCO3, 10 dextrose, 2.5 CaCl2, 2 M adenosine, saturated with 95% O2 and 5% CO2, pH 7.4, as previously [14]. Field excitatory postsynaptic.Error bars represent s.e.m. Table 1 Spine changes induced by NMDA receptor blockade, high Mg2+ and Stau1 siRNA treatment. thead th rowspan=”1″ colspan=”1″ /th th align=”center” colspan=”3″ rowspan=”1″ siRNA-CTL /th th align=”center” colspan=”3″ rowspan=”1″ siRNA-STAU1 /th th rowspan=”1″ colspan=”1″ /th th colspan=”6″ rowspan=”1″ hr / /th th rowspan=”1″ colspan=”1″ /th th align=”center” rowspan=”1″ colspan=”1″ Normal medium /th th align=”center” rowspan=”1″ colspan=”1″ Large Mg2+ (12 mM) /th th align=”center” rowspan=”1″ colspan=”1″ AP5 br / (100 M) /th th align=”center” rowspan=”1″ colspan=”1″ Normal medium /th th align=”center” rowspan=”1″ colspan=”1″ Large Mg2+ (12 mM) /th th align=”center” rowspan=”1″ colspan=”1″ AP5 br / (100 M) /th /thead Spine denseness (spine/m)0.29 0.020.21 0.02*0.34 0.030.28 0.020.19 0.020.34 0.03 hr / Spine length (m)1.15 0.021.3 0.02*1.3 0.02*1.29 0.021.35 0.031.32 0.03 hr / Spine shape br / (% of total) hr / Regular49.75 3.132.3 2.7*37.9 2.2*31.77 2.230.19 336.8 2.6 hr / Elongated40.92 2.354.68 2.3*50.3 2.3*51.49 251.35 2.753.1 2.7 hr / Filopodia9.33 1.513.02 1.911.8 1.616.74 1.415.57 310.2 1.5 Open in a separate window – Data indicated as mean s.e.m from 16-27 neurons per group, 4-8 indie experiments per group; in total 3129 protrusions were analyzed from 101 neurons. – * indicates significant difference in siRNA-CTL relative to normal medium; P 0.05, ANOVA. – indicates significant difference in siRNA-STAU1 relative to siRNA-CTL in individual condition; P 0.05, ANOVA. Although spine density was not affected by Stau1 down-regulation, spine length and shape were altered (Table ?(Table1).1). explained by these effects, since when tested in normal medium, slice cultures that had been treated with high Mg2+ (to impair NMDA receptor function) in combination with a control siRNA still exhibited late-LTP, while siRNA to Stau1 was still effective in obstructing late-LTP. Our results indicate that Stau1 involvement in spine morphogenesis is dependent on ongoing NMDA receptor-mediated plasticity, but its effects on late-LTP are self-employed of these changes. These findings clarify the part of Stau1-dependent mRNA rules in physiological and morphological changes underlying long-term synaptic plasticity in pyramidal cells. strong class=”kwd-title” Keywords: Schaffer security synapses, RNA transport, past due LTP, spontaneous activity-driven potentiation, spine morphogenesis Intro Localization of mRNAs to synaptic sites and their subsequent translation have emerged as important mechanisms contributing to synapse-specific plasticity [1,2]. Therefore, mRNA binding proteins (RBPs), which are key players in the transport of mRNAs, may be selectively implicated in various forms of plasticity that depend on the transport and local translation of specific transcripts. Staufen (Stau) [3,4], fragile mental retardation protein (FMRP) [5,6], zipcode-binding proteins [7] and cytoplasmic polyadenyation element binding protein (CPEB) [8,9] are RBPs known to be implicated in mRNA dendritic localization and translation in neurons. Notably, Stau is definitely implicated in rules of mRNAs required for memory space formation in Drosophila and Aplysia [10,11]. In mammals, the two members of the Stau family, Stau1 and Stau2, are present in unique ribonucleoprotein (RNP) complexes [12] and associate with different mRNAs [13]. Stau1 is required for the transport of mRNAs necessary for long-term potentiation at hippocampal synapses, as knockdown of Stau1 impaired dendritic transport of CaMKII mRNA in hippocampal neurons [3]. Moreover, downregulation of Stau1 also prevented the translation-dependent late phase LTP (late-LTP) induced by forskolin in CA1 pyramidal cells. However, the translation-independent early phase LTP was intact, suggesting an essential part of Stau1-dependent mRNA rules in protein synthesis associated with late-LTP [14]. Interestingly, we recently found that Stau2-dependent rules of mRNA was essential specifically for translation-dependent mGluR long-term major depression, uncovering selective mechanisms of mRNA rules for different forms of translation-dependent long-term synaptic plasticity [15]. Long-term changes in synaptic function are associated with changes in dendritic spines [16,17]. Indeed, we found that, in association with the impairment in late-LTP, Stau1 knockdown resulted in a shift from regular short spines to longer thin spines, suggesting a role in the formation and/or maintenance of mature spine shape [14]. However, since a form of NMDA-mediated plasticity, referred to as spontaneous activity-driven potentiation (SAP) [18], may be ongoing in our slice culture conditions and induce changes in spine shape [19-21], it is unknown whether the effects of Stau1 knockdown on late-LTP were due to its actions on spine morphogenesis, or vice versa. Thus, our aims were to test PQR309 directly if preventing SAP by blocking NMDAR function (or elevating extracellular Mg2+) would influence the changes in dendritic spine morphology induced by Stau1 knockdown, and whether the changes induced by blocking SAP were in turn required for the effect on Stau1 knockdown on late-LTP. We found that while Stau1 is usually involved in spine morphogenesis through NMDAR-mediated SAP, the change in spine morphogenesis was not important for the effect of Stau1 on late-LTP. Methods Organotypic hippocampal slice cultures All experiments were done in accordance with animal care guidelines at FLNC Universit de Montral, with the approval of the ethics committee at Universit de Montral (CDEA #10-003), and followed internationally recognized guidelines. Organotypic hippocampal slices were prepared and maintained in culture as previously described [14,22]. siRNAs and transfections siRNA target sequences for rat were as described [14]. Biolistic PQR309 transfection of neurons in organotypic slice cultures was performed using a Helios gene gun (Bio-Rad, CA) following manufacturer’s instructions as previously [14,22]. Electrophysiological recordings and cell imaging experiments were performed 48 hours after transfection.