Tabla espec. clave
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|B, M, R||ICC, IHC, WB||M||Purified||Monoclonal Antibody|
|Presentation||Purified immunoglobulin. Liquid in PBS, pH 7.2 with 50% glycerol and 0.09% sodium azide.|
|Safety Information according to GHS|
|Material Size||100 µg|
Ficha datos de seguridad (MSDS)
Referencias bibliográficas | 30 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Pub Med ID|
|STIM2 regulates PKA-dependent phosphorylation and trafficking of AMPARs. |
Garcia-Alvarez, G; Lu, B; Yap, KA; Wong, LC; Thevathasan, JV; Lim, L; Ji, F; Tan, KW; Mancuso, JJ; Tang, W; Poon, SY; Augustine, GJ; Fivaz, M
Molecular biology of the cell 26 1141-59 2015
STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca(2+) entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER-plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites.
|The X-linked mental retardation protein OPHN1 interacts with Homer1b/c to control spine endocytic zone positioning and expression of synaptic potentiation. |
Nakano-Kobayashi, A; Tai, Y; Nadif Kasri, N; Van Aelst, L
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 8665-71 2014
At glutamatergic synapses, local endocytic recycling of AMPA receptors (AMPARs) is important for the supply of a mobile pool of AMPARs required for synaptic potentiation. This local recycling of AMPARs critically relies on the presence of an endocytic zone (EZ) near the postsynaptic density (PSD). The precise mechanisms that couple the EZ to the PSD still remain largely elusive, with the large GTPase Dynamin-3 and the multimeric PSD adaptor protein Homer1 as the two main players identified. Here, we demonstrate that a physical interaction between the X-linked mental retardation protein oligophrenin-1 (OPHN1) and Homer1b/c is crucial for the positioning of the EZ adjacent to the PSD, and present evidence that this interaction is important for OPHN1's role in controlling activity-dependent strengthening of excitatory synapses in the rat hippocampus. Disruption of the OPHN1-Homer1b/c interaction causes a displacement of EZs from the PSD, along with impaired AMPAR recycling and reduced AMPAR accumulation at synapses, in both basal conditions and conditions that can induce synaptic potentiation. Together, our findings unveil a novel role for OPHN1 as an interaction partner of Homer1b/c in spine EZ positioning, and provide new mechanistic insight into how genetic deficits in OPHN1 can lead to impaired synapse maturation and plasticity.
|High content image analysis identifies novel regulators of synaptogenesis in a high-throughput RNAi screen of primary neurons. |
Nieland, TJ; Logan, DJ; Saulnier, J; Lam, D; Johnson, C; Root, DE; Carpenter, AE; Sabatini, BL
PloS one 9 e91744 2014
The formation of synapses, the specialized points of chemical communication between neurons, is a highly regulated developmental process fundamental to establishing normal brain circuitry. Perturbations of synapse formation and function causally contribute to human developmental and degenerative neuropsychiatric disorders, such as Alzheimer's disease, intellectual disability, and autism spectrum disorders. Many genes controlling synaptogenesis have been identified, but lack of facile experimental systems has made systematic discovery of regulators of synaptogenesis challenging. Thus, we created a high-throughput platform to study excitatory and inhibitory synapse development in primary neuronal cultures and used a lentiviral RNA interference library to identify novel regulators of synapse formation. This methodology is broadly applicable for high-throughput screening of genes and drugs that may rescue or improve synaptic dysfunction associated with cognitive function and neurological disorders.
|Postnatal expression of neurotrophic factors accessible to spiral ganglion neurons in the auditory system of adult hearing and deafened rats. |
Bailey, EM; Green, SH
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 13110-26 2014
Spiral ganglion neurons (SGNs) receive input from cochlear hair cells and project from the cochlea to the cochlear nucleus. After destruction of hair cells with aminoglycoside antibiotics or noise, SGNs gradually die. It has been assumed that SGN death is attributable to loss of neurotrophic factors (NTFs) derived from hair cells or supporting cells in the organ of Corti (OC). We used quantitative PCR (qPCR) to assay NTF expression-neurotrophin-3 (NT-3), BDNF, GDNF, neurturin, artemin, and CNTF-in the OC and cochlear nucleus at various ages from postnatal day 0 (P0) to P90 in control hearing and neonatally deafened rats. NT-3, neurturin, and CNTF were most abundant in the postnatal hearing OC; CNTF and neurturin most abundant in the cochlear nucleus. In the OC, NT-3 and CNTF showed a postnatal increase in expression approximately concomitant with hearing onset. In rats deafened by daily kanamycin injections (from P8 to P16), surviving inner hair cells were evident at P16 but absent by P19, with most postsynaptic boutons lost before P16. NT-3 and CNTF, which normally increase postnatally, had significantly reduced expression in the OC of deafened rats, although CNTF was expressed throughout the time that SGNs were dying. In contrast, neurturin expression was constant, unaffected by deafening or by age. CNTF and neurturin expression in the cochlear nucleus was unaffected by deafening or age. Thus, NTFs other than NT-3 are available to SGNs even as they are dying after deafening, apparently conflicting with the hypothesis that SGN death is attributable to lack of NTFs.
|Localization of diacylglycerol lipase alpha and monoacylglycerol lipase during postnatal development of the rat retina. |
Cécyre, B; Monette, M; Beudjekian, L; Casanova, C; Bouchard, JF
Frontiers in neuroanatomy 8 150 2014
In recent decades, there has been increased interest in the physiological roles of the endocannabinoid (eCB) system and its receptors, the cannabinoid receptor types 1 (CB1R) and 2 (CB2R). Exposure to cannabinoids during development results in neurofunctional alterations, which implies that the eCB system is involved in the developmental processes of the brain. Because of their lipophilic nature, eCBs are synthesized on demand and are not stored in vesicles. Consequently, the enzymes responsible for their synthesis and degradation are key regulators of their physiological actions. Therefore, knowing the localization of these enzymes during development is crucial for a better understanding of the role played by eCBs during the formation of the central nervous system. In this study, we investigated the developmental protein localization of the synthesizing and catabolic enzymes of the principal eCB, 2-arachidonoylglycerol (2-AG) in the retinas of young and adult rats. The distribution of the enzymes responsible for the synthesis (DAGLα) and the degradation (MAGL) of 2-AG was determined for every retinal cell type from birth to adulthood. Our results indicate that DAGLα is present early in postnatal development. It is highly expressed in photoreceptor, horizontal, amacrine, and ganglion cells. MAGL appears later during the development of the retina and its presence is limited to amacrine and Müller cells. Overall, these results suggest that 2-AG is strongly present in early retinal development and might be involved in the regulation of the structural and functional maturation of the retina.
|Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons. |
Galic, M; Tsai, FC; Collins, SR; Matis, M; Bandara, S; Meyer, T
eLife 3 e03116 2014
In the vertebrate central nervous system, exploratory filopodia transiently form on dendritic branches to sample the neuronal environment and initiate new trans-neuronal contacts. While much is known about the molecules that control filopodia extension and subsequent maturation into functional synapses, the mechanisms that regulate initiation of these dynamic, actin-rich structures have remained elusive. Here, we find that filopodia initiation is suppressed by recruitment of ArhGAP44 to actin-patches that seed filopodia. Recruitment is mediated by binding of a membrane curvature-sensing ArhGAP44 N-BAR domain to plasma membrane sections that were deformed inward by acto-myosin mediated contractile forces. A GAP domain in ArhGAP44 triggers local Rac-GTP hydrolysis, thus reducing actin polymerization required for filopodia formation. Additionally, ArhGAP44 expression increases during neuronal development, concurrent with a decrease in the rate of filopodia formation. Together, our data reveals a local auto-regulatory mechanism that limits initiation of filopodia via protein recruitment to nanoscale membrane deformations.
|Ribbon synapse plasticity in the cochleae of Guinea pigs after noise-induced silent damage. |
Shi, L; Liu, L; He, T; Guo, X; Yu, Z; Yin, S; Wang, J
PloS one 8 e81566 2013
Noise exposure at low levels or low doses can damage hair cell afferent ribbon synapses without causing permanent threshold shifts. In contrast to reports in the mouse cochleae, initial damage to ribbon synapses in the cochleae of guinea pigs is largely repairable. In the present study, we further investigated the repair process in ribbon synapses in guinea pigs after similar noise exposure. In the control samples, a small portion of afferent synapses lacked synaptic ribbons, suggesting the co-existence of conventional no-ribbon and ribbon synapses. The loss and recovery of hair cell ribbons and post-synaptic densities (PSDs) occurred in parallel, but the recovery was not complete, resulting in a permanent loss of less than 10% synapses. During the repair process, ribbons were temporally separated from the PSDs. A plastic interaction between ribbons and postsynaptic terminals may be involved in the reestablishment of synaptic contact between ribbons and PSDs, as shown by location changes in both structures. Synapse repair was associated with a breakdown in temporal processing, as reflected by poorer responses in the compound action potential (CAP) of auditory nerves to time-stress signals. Thus, deterioration in temporal processing originated from the cochlea. This deterioration developed with the recovery in hearing threshold and ribbon synapse counts, suggesting that the repaired synapses had deficits in temporal processing.
|An in vitro model of developmental synaptogenesis using cocultures of human neural progenitors and cochlear explants. |
Nayagam, BA; Edge, AS; Needham, K; Hyakumura, T; Leung, J; Nayagam, DA; Dottori, M
Stem cells and development 22 901-12 2013
In mammals, the sensory hair cells and auditory neurons do not spontaneously regenerate and their loss results in permanent hearing impairment. Stem cell therapy is one emerging strategy that is being investigated to overcome the loss of sensory cells after hearing loss. To successfully replace auditory neurons, stem cell-derived neurons must be electrically active, capable of organized outgrowth of processes, and of making functional connections with appropriate tissues. We have developed an in vitro assay to test these parameters using cocultures of developing cochlear explants together with neural progenitors derived from human embryonic stem cells (hESCs). We found that these neural progenitors are electrically active and extend their neurites toward the sensory hair cells in cochlear explants. Importantly, this neurite extension was found to be significantly greater when neural progenitors were predifferentiated toward a neural crest-like lineage. When grown in coculture with hair cells only (denervated cochlear explants), stem cell-derived processes were capable of locating and growing along the hair cell rows in an en passant-like manner. Many presynaptic terminals (synapsin 1-positive) were observed between hair cells and stem cell-derived processes in vitro. These results suggest that differentiated hESC-derived neural progenitors may be useful for developing therapies directed at auditory nerve replacement, including complementing emerging hair cell regeneration therapies.
|Expression of voltage-gated calcium channel α(2)δ(4) subunits in the mouse and rat retina. |
De Sevilla Müller, LP; Liu, J; Solomon, A; Rodriguez, A; Brecha, NC
The Journal of comparative neurology 521 2486-501 2013
High-voltage activated Ca channels participate in multiple cellular functions, including transmitter release, excitation, and gene transcription. Ca channels are heteromeric proteins consisting of a pore-forming α(1) subunit and auxiliary α(2)δ and β subunits. Although there are reports of α(2)δ(4) subunit mRNA in the mouse retina and localization of the α(2)δ(4) subunit immunoreactivity to salamander photoreceptor terminals, there is a limited overall understanding of its expression and localization in the retina. α(2)δ(4) subunit expression and distribution in the mouse and rat retina were evaluated by using reverse transcriptase polymerase chain reaction, western blot, and immunohistochemistry with specific primers and a well-characterized antibody to the α(2)δ(4) subunit. α(2)δ(4) subunit mRNA and protein are present in mouse and rat retina, brain, and liver homogenates. Immunostaining for the α(2)δ(4) subunit is mainly localized to Müller cell processes and endfeet, photoreceptor terminals, and photoreceptor outer segments. This subunit is also expressed in a few displaced ganglion cells and bipolar cell dendrites. These findings suggest that the α(2)δ(4) subunit participates in the modulation of L-type Ca(2+) current regulating neurotransmitter release from photoreceptor terminals and Ca(2+)-dependent signaling pathways in bipolar and Müller cells.
|Low density lipoprotein receptor-related protein 1 (LRP1) modulates N-methyl-D-aspartate (NMDA) receptor-dependent intracellular signaling and NMDA-induced regulation of postsynaptic protein complexes. |
Nakajima, C; Kulik, A; Frotscher, M; Herz, J; Schäfer, M; Bock, HH; May, P
The Journal of biological chemistry 288 21909-23 2013
The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes γ-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1.
|Synaptic and extrasynaptic location of the receptor tyrosine kinase met during postnatal development in the mouse neocortex and hippocampus. |
Eagleson, KL; Milner, TA; Xie, Z; Levitt, P
The Journal of comparative neurology 521 3241-59 2013
MET, a replicated autism risk gene, encodes a pleiotropic receptor tyrosine kinase implicated in multiple cellular processes during development and following injury. Previous studies suggest that Met modulates excitatory synapse development in the neocortex and hippocampus, although the underlying mechanism is unknown. The peak of Met expression corresponds to the period of process outgrowth and synaptogenesis, with robust expression in hippocampal and neocortical neuropil. Resolving whether neuropil expression represents presynaptic, postsynaptic or glial localization provides insight into potential mechanisms of Met action. The subcellular distribution of Met was characterized using complementary ultrastructural, in situ proximity ligation assay (PLA), and biochemical approaches. At postnatal day (P) 7, immunoelectron microscopy revealed near-equivalent proportions of Met-immunoreactive pre- (axons and terminals) and postsynaptic (dendritic shafts and spines) profiles in the stratum radiatum in the hippocampal CA1 region. Staining was typically in elements in which the corresponding pre- or postsynaptic apposition was unlabeled. By P21, Met-immunoreactive presynaptic profiles predominated and ~20% of Met-expressing profiles were glial. A different distribution of Met-immunoreactive profiles was observed in layer V of somatosensory cortex: Met-labeled spines were rare and a smaller proportion of glial profiles expressed Met. Strikingly, Met-immunoreactive presynaptic profiles predominated over postsynaptic profiles as early as P7. PLA analysis of neurons in vitro and biochemical analysis of tissue subsynaptic fractions confirmed the localization of Met in specific synaptic subcompartments. The study demonstrates that Met is enriched at synapses during development and its activation may modulate synapse formation and stability through both pre- and postsynaptic mechanisms.
|Dynein interacts with the neural cell adhesion molecule (NCAM180) to tether dynamic microtubules and maintain synaptic density in cortical neurons. |
Perlson, E; Hendricks, AG; Lazarus, JE; Ben-Yaakov, K; Gradus, T; Tokito, M; Holzbaur, EL
The Journal of biological chemistry 288 27812-24 2013
Cytoplasmic dynein is well characterized as an organelle motor, but dynein also acts to tether and stabilize dynamic microtubule plus-ends in vitro. Here we identify a novel and direct interaction between dynein and the 180-kDa isoform of the neural cell adhesion molecule (NCAM). Optical trapping experiments indicate that dynein bound to beads via the NCAM180 interaction domain can tether projecting microtubule plus-ends. Live cell assays indicate that the NCAM180-dependent recruitment of dynein to the cortex leads to the selective stabilization of microtubules projecting to NCAM180 patches at the cell periphery. The dynein-NCAM180 interaction also enhances cell-cell adhesion in heterologous cell assays. Dynein and NCAM180 co-precipitate from mouse brain extract and from synaptosomal fractions, consistent with an endogenous interaction in neurons. Thus, we examined microtubule dynamics and synaptic density in primary cortical neurons. We find that depletion of NCAM, inhibition of the dynein-NCAM180 interaction, or dampening of microtubule dynamics with low dose nocodazole all result in significantly decreased in synaptic density. Based on these observations, we propose a working model for the role of dynein at the synapse, in which the anchoring of the motor to the cortex via binding to an adhesion molecule mediates the tethering of dynamic microtubule plus-ends to potentiate synaptic stabilization.
|Adaptor protein APPL1 couples synaptic NMDA receptor with neuronal prosurvival phosphatidylinositol 3-kinase/Akt pathway. |
Wang, YB; Wang, JJ; Wang, SH; Liu, SS; Cao, JY; Li, XM; Qiu, S; Luo, JH
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 11919-29 2012
It is well known that NMDA receptors (NMDARs) can both induce neurotoxicity and promote neuronal survival under different circumstances. Recent studies show that such paradoxical responses are related to the receptor location: the former to the extrasynaptic and the latter to the synaptic. The phosphoinositide 3-kinase (PI3K)/Akt kinase cascade is a key pathway responsible for the synaptic NMDAR-dependent neuroprotection. However, it is still unknown how synaptic NMDARs are coupled with the PI3K/Akt pathway. Here, we explored the role of an adaptor protein-adaptor protein containing pH domain, PTB domain, and leucine zipper motif (APPL1)-in this signal coupling using rat cortical neurons. We found that APPL1 existed in postsynaptic densities and associated with the NMDAR complex through binding to PSD95 at its C-terminal PDZ-binding motif. NMDARs, APPL1, and the PI3K/Akt cascade formed a complex in rat cortical neurons. Synaptic NMDAR activity increased the association of this complex, induced activation of the PI3K/Akt pathway, and consequently protected neurons against starvation-induced apoptosis. Perturbing APPL1 interaction with PSD95 by a peptide comprising the APPL1 C-terminal PDZ-binding motif dissociated the PI3K/Akt pathway from NMDARs. Either the peptide or lentiviral knockdown of APPL1 blocked synaptic NMDAR-dependent recruitment and activation of PI3K/Akt pathway, and consequently blocked synaptic NMDAR-dependent neuroprotection. These results suggest that APPL1 contributes to connecting synaptic NMDARs with the intracellular PI3K/Akt cascade and the downstream prosurvival signaling pathway in rat cortical neurons.
|Genetic deletion of NR3A accelerates glutamatergic synapse maturation. |
Henson, MA; Larsen, RS; Lawson, SN; Pérez-Otaño, I; Nakanishi, N; Lipton, SA; Philpot, BD
PloS one 7 e42327 2012
Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth.
|Molecular and functional interaction between protocadherin-γC5 and GABAA receptors. |
Li, Y; Xiao, H; Chiou, TT; Jin, H; Bonhomme, B; Miralles, CP; Pinal, N; Ali, R; Chen, WV; Maniatis, T; De Blas, AL
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 11780-97 2012
We have found that the γ2 subunit of the GABA(A) receptor (γ2-GABA(A)R) specifically interacts with protocadherin-γC5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large intracellular loop of the γ2-GABA(A)R and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 coimmunoprecipitates with GABA(A)Rs. In cotransfected HEK293 cells, Pcdh-γC5 promotes the transfer of γ2-GABA(A)R to the cell surface. We have previously shown that, in cultured hippocampal neurons, endogenous Pcdh-γC5 forms clusters, some of which associate with GABAergic synapses. Overexpression of Pcdh-γC5 in hippocampal neurons increases the density of γ2-GABA(A)R clusters but has no significant effect on the number of GABAergic contacts that these neurons receive, indicating that Pcdh-γC5 is not synaptogenic. Deletion of the cytoplasmic domain of Pcdh-γC5 enhanced its surface expression but decreased the association with both γ2-GABA(A)R clusters and presynaptic GABAergic contacts. Cultured hippocampal neurons from the Pcdh-γ triple C-type isoform knock-out (TCKO) mouse (Pcdhg(tcko/tcko)) showed plenty of GABAergic synaptic contacts, although their density was reduced compared with sister cultures from wild-type and heterozygous mice. Knocking down Pcdh-γC5 expression with shRNA decreased γ2-GABA(A)R cluster density and GABAergic innervation. The results indicate that, although Pcdh-γC5 is not essential for GABAergic synapse formation or GABA(A)R clustering, (1) Pcdh-γC5 regulates the surface expression of GABA(A)Rs via cis-cytoplasmic interaction with γ2-GABA(A)R, and (2) Pcdh-γC5 plays a role in the stabilization and maintenance of some GABAergic synapses.
|Genetic deletion of TNF receptor suppresses excitatory synaptic transmission via reducing AMPA receptor synaptic localization in cortical neurons. |
He, P; Liu, Q; Wu, J; Shen, Y
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 26 334-45 2012
The distribution of postsynaptic glutamate receptors has been shown to be regulated by proimmunocytokine tumor necrosis factor α (TNF-α) signaling. The role of TNF-α receptor subtypes in mediating glutamate receptor expression, trafficking, and function still remains unclear. Here, we report that TNF receptor subtypes (TNFR1 and TNFR2) differentially modulate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) clustering and function in cultured cortical neurons. We find that genetic deletion of TNFR1 decreases surface expression and synaptic localization of the AMPAR GluA1 subunit, reduces the frequency of miniature excitatory postsynaptic current (mEPSC), and reduces AMPA-induced maximal whole-cell current. In addition, these results are not observed in TNFR2-deleted neurons. The decreased AMPAR expression and function in TNFR1-deleted cells are not significantly restored by short (2 h) or long (24 h) term exposure to TNF-α. In TNFR2-deleted cells, TNF-α promotes AMPAR trafficking to the synapse and increases mEPSC frequency. In the present study, we find no significant change in the GluN1 subunit of NMDAR clusters, location, and mEPSC. This includes applying or withholding the TNF-α treatment in both TNFR1- and TNFR2-deleted neurons. Our results indicate that TNF receptor subtype 1 but not 2 plays a critical role in modulating AMPAR clustering, suggesting that targeting TNFR1 gene might be a novel approach to preventing neuronal AMPAR-mediated excitotoxicity.
|Dysregulation of BDNF-TrkB signaling in developing hippocampal neurons by Pb(2+): implications for an environmental basis of neurodevelopmental disorders. |
Stansfield, KH; Pilsner, JR; Lu, Q; Wright, RO; Guilarte, TR
Toxicological sciences : an official journal of the Society of Toxicology 127 277-95 2012
Dysregulation of synaptic development and function has been implicated in the pathophysiology of neurodegenerative disorders and mental disease. A neurotrophin that has an important function in neuronal and synaptic development is brain-derived neurotrophic factor (BDNF). In this communication, we examined the effects of lead (Pb(2+)) exposure on BDNF-tropomyosin-related kinase B (TrkB) signaling during the period of synaptogenesis in cultured neurons derived from embryonic rat hippocampi. We show that Pb(2+) exposure decreases BDNF gene and protein expression, and it may also alter the transport of BDNF vesicles to sites of release by altering Huntingtin phosphorylation and protein levels. Combined, these effects of Pb(2+) resulted in decreased concentrations of extracellular mature BDNF. The effect of Pb(2+) on BDNF gene expression was associated with a specific decrease in calcium-sensitive exon IV transcript levels and reduced phosphorylation and protein expression of the transcriptional repressor methyl-CpG-binding protein (MeCP2). TrkB protein levels and autophosphorylation at tyrosine 816 were significantly decreased by Pb(2+) exposure with a concomitant increase in p75 neurotrophin receptor (p75(NTR)) levels and altered TrkB-p75(NTR) colocalization. Finally, phosphorylation of Synapsin I, a presynaptic target of BDNF-TrkB signaling, was significantly decreased by Pb(2+) exposure with no effect on total Synapsin I protein levels. This effect of Pb(2+) exposure on Synapsin I phosphorylation may help explain the impairment in vesicular release documented by us previously (Neal, A. P., Stansfield, K. H., Worley, P. F., Thompson, R. E., and Guilarte, T. R. (2010). Lead exposure during synaptogenesis alters vesicular proteins and impairs vesicular release: Potential role of N-Methyl-D-aspartate receptor (NMDAR) dependent BDNF signaling. Toxicol. Sci. 116, 249-263) because it controls vesicle movement from the reserve pool to the readily releasable pool. In summary, the present study demonstrates that Pb(2+) exposure during the period of synaptogenesis of hippocampal neurons in culture disrupts multiple synaptic processes regulated by BDNF-TrkB signaling with long-term consequences for synaptic function and neuronal development.
|Rescue of the genetically engineered Cul4b mutant mouse as a potential model for human X-linked mental retardation. |
Chen, CY; Tsai, MS; Lin, CY; Yu, IS; Chen, YT; Lin, SR; Juan, LW; Chen, YT; Hsu, HM; Lee, LJ; Lin, SW
Human molecular genetics 21 4270-85 2012
Mutation in CUL4B, which encodes a scaffold protein of the E3 ubiquitin ligase complex, has been found in patients with X-linked mental retardation (XLMR). However, early deletion of Cul4b in mice causes prenatal lethality, which has frustrated attempts to characterize the phenotypes in vivo. In this report, we successfully rescued Cul4b mutant mice by crossing female mice in which exons 4-5 of Cul4b were flanked by loxP sequences with Sox2-Cre male mice. In Cul4b-deficient (Cul4b(Δ)/Y) mice, no CUL4B protein was detected in any of the major organs, including the brain. In the hippocampus, the levels of CUL4A, CUL4B substrates (TOP1, β-catenin, cyclin E and WDR5) and neuronal markers (MAP2, tau-1, GAP-43, PSD95 and syn-1) were not sensitive to Cul4b deletion, whereas the number of parvalbumin (PV)-positive GABAergic interneurons was decreased in Cul4b(Δ)/Y mice, especially in the dentate gyrus (DG). Some dendritic features, including the complexity, diameter and spine density in the CA1 and DG hippocampal neurons, were also affected by Cul4b deletion. Together, the decrease in the number of PV-positive neurons and altered dendritic properties in Cul4b(Δ)/Y mice imply a reduction in inhibitory regulation and dendritic integration in the hippocampal neural circuit, which lead to increased epileptic susceptibility and spatial learning deficits. Our results identify Cul4b(Δ)/Y mice as a potential model for the non-syndromic model of XLMR that replicates the CUL4B-associated MR and is valuable for the development of a therapeutic strategy for treating MR.
|Preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient. |
Pamela R Westmark,Cara J Westmark,Athavi Jeevananthan,James S Malter
Journal of visualized experiments : JoVE 2011
Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters. The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.
|Reduced cortical BDNF expression and aberrant memory in Carf knock-out mice. |
McDowell, KA; Hutchinson, AN; Wong-Goodrich, SJ; Presby, MM; Su, D; Rodriguiz, RM; Law, KC; Williams, CL; Wetsel, WC; West, AE
The Journal of neuroscience : the official journal of the Society for Neuroscience 30 7453-65 2010
Transcription factors are a key point of convergence between the cell-intrinsic and extracellular signals that guide synaptic development and brain plasticity. Calcium-response factor (CaRF) is a unique transcription factor first identified as a binding protein for a calcium-response element in the gene encoding brain-derived neurotrophic factor (Bdnf). We have now generated Carf knock-out (KO) mice to characterize the function of this factor in vivo. Intriguingly, Carf KO mice have selectively reduced expression of Bdnf exon IV-containing mRNA transcripts and BDNF protein in the cerebral cortex, whereas BDNF levels in the hippocampus and striatum remain unchanged, implicating CaRF as a brain region-selective regulator of BDNF expression. At the cellular level, Carf KO mice show altered expression of GABAergic proteins at striatal synapses, raising the possibility that CaRF may contribute to aspects of inhibitory synapse development. Carf KO mice show normal spatial learning in the Morris water maze and normal context-dependent fear conditioning. However they have an enhanced ability to find a new platform location on the first day of reversal training in the water maze and they extinguish conditioned fear more slowly than their wild-type littermates. Finally, Carf KO mice show normal short-term (STM) and long-term memory (LTM) in a novel object recognition task, but exhibit impairments during the remote memory phase of testing. Together, these data reveal novel roles for CaRF in the organization and/or function of neural circuits that underlie essential aspects of learning and memory.Artículo Texto completo
|Postnatal and ovariectomic regulation of postsynaptic density protein-95 in the hippocampus of female Sprague-Dawley rats. |
Zhang D, Zhang J, Bian C, Deng Q
Synapse 64 875-8. 2010
Postsynaptic density protein-95 (PSD-95) is hypothesized to control the excitatory-to-inhibitory ratio and plays an important role in the regulation of hippocampal synaptic plasticity, synaptogenesis, and learning and memory. In this report, we used immunoblotting to study the effects of aging and ovariectomy (OVX) on the expression of PSD-95 in the hippocampus of female rats. The results indicated that postnatal expression of hippocampal PSD-95 correlated with the fluctuation of circulating female sex hormones such as estrogen. Neonatal PSD-95 level was very low, but dramatically increased within the first month. The highest expression of PSD-95 was detected at postnatal day 30 (P30) and significantly decreased by 18 months. In the adult hippocampus, OVX significantly decreased PSD-95 expression within the first week, but it had recovered to adult levels 2 weeks later. Taken together, we conclude that circulating ovarian hormones may play a crucial role in the regulation of excitatory synapses within the hippocampus. Depletion of ovarian hormones can transiently and dramatically decrease the level of excitatory synapses for a limited time.
|Synaptic and nonsynaptic localization of protocadherin-gammaC5 in the rat brain. |
Li, Y; Serwanski, DR; Miralles, CP; Fiondella, CG; Loturco, JJ; Rubio, ME; De Blas, AL
The Journal of comparative neurology 518 3439-63 2010
It has been proposed that gamma-protocadherins (Pcdh-gammas) are involved in the establishment of specific patterns of neuronal connectivity. Contrary to the other Pcdh-gammas, which are expressed in the embryo, Pcdh-gammaC5 is expressed postnatally in the brain, coinciding with the peak of synaptogenesis. We have developed an antibody specific for Pcdh-gammaC5 to study the expression and localization of Pcdh-gammaC5 in brain. Pcdh-gammaC5 is highly expressed in the olfactory bulb, corpus striatum, dentate gyrus, CA1 region of the hippocampus, layers I and II of the cerebral cortex, and molecular layer of the cerebellum. Pcdh-gammaC5 is expressed in both neurons and astrocytes. In hippocampal neuronal cultures, and in the absence of astrocytes, a significant percentage of synapses, more GABAergic than glutamatergic, have associated Pcdh-gammaC5 clusters. Some GABAergic axons show Pcdh-gammaC5 in the majority of their synapses. Nevertheless, many Pcdh-gammaC5 clusters are not associated with synapses. In the brain, significant numbers of Pcdh-gammaC5 clusters are located at contact points between neurons and astrocytes. Electron microscopic immunocytochemistry of the rat brain shows that 1) Pcdh-gammaC5 is present in some GABAergic and glutamatergic synapses both pre- and postsynaptically; 2) Pcdh-gammaC5 is also extrasynaptically localized in membranes and in cytoplasmic organelles of neurons and astrocytes; and 3) Pcdh-gammaC5 is also localized in perisynaptic astrocyte processes. The results support the notions that 1) Pcdh-gammaC5 plays a role in synaptic specificity and/or synaptic maturation and 2) Pcdh-gammaC5 is involved in neuron-neuron synaptic interactions and in neuron-astrocyte interactions, including perisynaptic neuron-astrocyte interactions.
|Transcriptome analysis of synaptoneurosomes identifies neuroplasticity genes overexpressed in incipient Alzheimer's disease. |
Williams, C; Mehrian Shai, R; Wu, Y; Hsu, YH; Sitzer, T; Spann, B; McCleary, C; Mo, Y; Miller, CA
PloS one 4 e4936 2009
In Alzheimer's disease (AD), early deficits in learning and memory are a consequence of synaptic modification induced by toxic beta-amyloid oligomers (oAbeta). To identify immediate molecular targets downstream of oAbeta binding, we prepared synaptoneurosomes from prefrontal cortex of control and incipient AD (IAD) patients, and isolated mRNAs for comparison of gene expression. This novel approach concentrates synaptic mRNA, thereby increasing the ratio of synaptic to somal mRNA and allowing discrimination of expression changes in synaptically localized genes. In IAD patients, global measures of cognition declined with increasing levels of dimeric Abeta (dAbeta). These patients also showed increased expression of neuroplasticity related genes, many encoding 3'UTR consensus sequences that regulate translation in the synapse. An increase in mRNA encoding the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) was paralleled by elevated expression of the corresponding protein in IAD. These results imply a functional impact on synaptic transmission as GluR2, if inserted, maintains the receptors in a low conductance state. Some overexpressed genes may induce early deficits in cognition and others compensatory mechanisms, providing targets for intervention to moderate the response to dAbeta.
|Cold-induced exodus of postsynaptic proteins from dendritic spines. |
Hui-Hsuan Cheng, Zu-Han Huang, Wei-Hsiang Lin, Wei-Yuan Chow, Yen-Chung Chang, Hui-Hsuan Cheng, Zu-Han Huang, Wei-Hsiang Lin, Wei-Yuan Chow, Yen-Chung Chang, Hui-Hsuan Cheng, Zu-Han Huang, Wei-Hsiang Lin, Wei-Yuan Chow, Yen-Chung Chang
Journal of neuroscience research 87 460-9 2009
Dendritic spines are small protrusions on neuronal dendrites and the major target of the excitatory inputs in mammalian brains. Cultured neurons and brain slices are important tools in studying the biochemical and cellular properties of dendritic spines. During the processes of immunocytochemical studies of neurons and the preparation of brain slices, neurons were often kept at temperatures lower than 37 degrees C for varied lengths of time. This study sought to investigate whether and how cold treatment would affect the protein composition of dendritic spines. The results indicated that upon cold treatment four postsynaptic proteins, namely, alpha,beta-tubulins, calcium, calmodulin-dependent protein kinase IIalpha, and cytoplasmic dynein heavy chain and microtubule-associated protein 2, but not PSD-95 or AMPA receptors, exited from the majority of dendritic spines of cultured rat hippocampal neurons in a Gd(3+)-sensitive manner. The cold-induced exit of tubulins from dendritic spines was further found to be an energy-dependent process involving the activation of Gd(3+)-sensitive calcium channels and ryanodine receptors. The results thus indicate that changes in temperature, calcium concentration, and energy supply of the medium surrounding neurons would affect the protein composition of the dendritic spines and conceivably the protein composition of the subcellular organizations, such as the postsynaptic density, in the cytoplasm of dendritic spines.
|Awake intranasal insulin delivery modifies protein complexes and alters memory, anxiety, and olfactory behaviors. |
Marks, DR; Tucker, K; Cavallin, MA; Mast, TG; Fadool, DA
The Journal of neuroscience : the official journal of the Society for Neuroscience 29 6734-51 2009
The role of insulin pathways in olfaction is of significant interest with the widespread pathology of diabetes mellitus and its associated metabolic and neuronal comorbidities. The insulin receptor (IR) kinase is expressed at high levels in the olfactory bulb, in which it suppresses a dominant Shaker ion channel (Kv1.3) via tyrosine phosphorylation of critical N- and C-terminal residues. We optimized a 7 d intranasal insulin delivery (IND) in awake mice to ascertain the biochemical and behavioral effects of insulin to this brain region, given that nasal sprays for insulin have been marketed notwithstanding our knowledge of the role of Kv1.3 in olfaction, metabolism, and axon targeting. IND evoked robust phosphorylation of Kv1.3, as well as increased channel protein-protein interactions with IR and postsynaptic density 95. IND-treated mice had an increased short- and long-term object memory recognition, increased anxiolytic behavior, and an increased odor discrimination using an odor habituation protocol but only moderate change in odor threshold using a two-choice paradigm. Unlike Kv1.3 gene-targeted deletion that alters metabolism, adiposity, and axonal targeting to defined olfactory glomeruli, suppression of Kv1.3 via IND had no effect on body weight nor the size and number of M72 glomeruli or the route of its sensory axon projections. There was no evidence of altered expression of sensory neurons in the epithelium. In mice made prediabetic via diet-induced obesity, IND was no longer effective in increasing long-term object memory recognition nor increasing anxiolytic behavior, suggesting state dependency or a degree of insulin resistance related to these behaviors.
|Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retina. |
Heather D VanGuilder,Robert M Brucklacher,Kruti Patel,Rhona W Ellis,Willard M Freeman,Alistair J Barber
The European journal of neuroscience 28 2008
Diabetic retinopathy can result in vision loss and involves progressive neurovascular degeneration of the retina. This study tested the hypothesis that diabetes decreases the retinal expression of presynaptic proteins involved in synaptic function. The protein and mRNA contents for synapsin I, synaptophysin, vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa and postsynaptic density protein of 95 kDa were measured by immunohistochemistry, immunoblotting and real-time quantitative polymerase chain reaction in whole retinas and retinal synaptosomes from streptozotocin-diabetic and control Sprague-Dawley rats. There was less presynaptic protein immunoreactivity after 1 and 3 months of diabetes than in controls. Discrete synaptophysin-immunoreactive puncta were significantly smaller and fewer in sections from 1- and 3-month diabetic rat retinas than in those from controls. The content of presynaptic proteins was significantly less in whole retinas of 1- and 3-month diabetic rats, and in synaptosomes from 1-month diabetic rats, than in controls. Whole retinas had significantly less mRNA for these genes after 3 months but not 1 month of diabetes, as compared to controls (with the exception of postsynaptic density protein of 95 kDa). In contrast, there was significantly less mRNA for synaptic proteins in synaptosomes of 1-month diabetic rats than in controls, suggesting a localized depletion at synapses. Protein and mRNA for beta-actin and neuron-specific enolase were unchanged by diabetes. The ratio of phosphorylated to total synapsin I was also reduced in whole retina and isolated synaptosomes from 1-month diabetic rats, as compared to controls. These data suggest that diabetes has a profound impact on presynaptic protein expression in the retina, and may provide a mechanism for the well-established defects in vision and the electrophysiological response of the retina in diabetes.
|Telencephalic neurons monosynaptically link brainstem and forebrain premotor networks necessary for song. |
Roberts, TF; Klein, ME; Kubke, MF; Wild, JM; Mooney, R
The Journal of neuroscience : the official journal of the Society for Neuroscience 28 3479-89 2008
Birdsong, like human speech, is a series of learned vocal gestures resulting from the coordination of vocal and respiratory brainstem networks under the control of the telencephalon. The song motor circuit includes premotor and motor cortical analogs, known as HVC (used as a proper name) and RA (the robust nucleus of the arcopallium), respectively. Previous studies showed that HVC projects to RA and that RA projection neurons (PNs) topographically innervate brainstem vocal-motor and respiratory networks. The idea that singing-related activity flows between HVC and RA in a strictly feedforward manner is a central component of all models of song production. In contrast to this prevailing view of song motor circuit organization, we show that RA sends a reciprocal projection directly to HVC. Lentiviral labeling of RA PN axons and transgene tagging of RA PN synaptic terminals reveal a direct projection from RA to HVC. Retrograde tracing from HVC demonstrates that this projection originates exclusively from neurons in dorsocaudal regions of RA. Using dual retrograde tracer injections, we further show that many of these RA(HVC) neurons also innervate the brainstem nucleus retroambigualis, which is premotor to expiratory motoneurons, thereby identifying a population of RA PNs positioned to coordinate activity at higher and lower levels of the song motor circuit. In combination, our findings identify a previously unknown pathway that may enable a subset of RA neurons to provide song-related signals to the respiratory brainstem but also transmit a copy of this information to song patterning networks in HVC.
|Immunoautoradiographic analysis of NMDA receptor subunits and associated postsynaptic density proteins in the brain of dyskinetic MPTP-treated common marmosets. |
Hurley, M J, et al.
Eur. J. Neurosci., 21: 3240-50 (2005) 2005
l-3,4-dihydroxyphenylalanine methyl ester (l-DOPA)-induced dyskinesia in Parkinson's disease may result from aberrant glutamatergic stimulation of the striatum due to synaptic plasticity in the motor cortex or striatum as a consequence of adaptation of striatal output pathways. This might result from changes in NMDA receptor subunit or NMDA receptor associated postsynaptic density (PSD) scaffold protein expression. Using immunoautoradiography the expression levels of NR1 and NR2B subunits of the NMDA receptor and the postsynaptic density scaffold proteins, PSD-95, PSD-93, and neurofilament light (NFL) were examined in normal common marmosets (Callithrix jacchus) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned animals that exhibited high or low levels of l-DOPA-induced dyskinesia. Brains from MPTP-lesioned animals that were not primed for l-DOPA-induced dyskinesia were not included in this study. No alterations in the NR1 NMDA receptor subunit were observed. The NR2B NMDA receptor subunit was increased in caudal caudate nucleus and putamen, hippocampus, cingulate motor area (CMA), supplementary motor area (SMA) and dorsal primary motor cortex (dMI) of highly dyskinetic MPTP-lesioned marmosets, but not in animals with low levels of dyskinesia. PSD-93 was decreased in the globus pallidus of marmosets with high and low levels of dyskinesia and increased in the CMA, SMA and dMI of highly dyskinetic marmosets. PSD-95 was increased in the SMA of highly dyskinetic marmosets, but not in animals with low dyskinesia. NFL expression was elevated in the SMA and dorsal and ventral MI of highly dyskinetic marmosets. These results suggest that l-DOPA treatment of MPTP-lesioned marmosets can affect glutamatergic systems and indicate that altered NMDA receptor function may relate to dyskinesia.
|Up-regulation of peripherin is associated with alterations in synaptic plasticity in CA1 and CA3 regions of hippocampus. |
Jasna Kriz, Jean-Martin Beaulieu, Jean-Pierre Julien, Kresimir Krnjević
Neurobiology of disease 18 409-20 2005
Peripherin is a type III intermediate filament protein normally undetectable in most brain neurons. Here, we report a similar pattern of peripherin expression in the brains of both mice treated with systemic injections of kainic acid (KA) and in peripherin transgenic mice (Per mice) over-expressing the normal peripherin gene under its own promoter. Double-immunofluorescence labeling revealed a partial co-localization of peripherin with the microtubule-associated protein MAP2, but not with neurofilament proteins. Electrophysiological studies revealed that synaptic plasticity was markedly altered in Per mice: in CA1, long-term potentiation (LTP) was decreased in Per slices (+29 +/- 2.0%, vs. +58 +/- 5.4%, in WT); while in CA3, LTP was increased in Per (+63 +/- 3.5% vs. +43 +/- 2.4.0%). In the hippocampus of Per mice, the levels of MAP2 were decreased, though synaptophysin and PSD95 remained unchanged. These intriguing findings suggest a role of peripherin in the alteration of hippocampal synaptic plasticity.
|Visual deprivation modifies both presynaptic glutamate release and the composition of perisynaptic/extrasynaptic NMDA receptors in adult visual cortex. |
Yashiro, K; Corlew, R; Philpot, BD
The Journal of neuroscience : the official journal of the Society for Neuroscience 25 11684-92 2005
Use-dependent modifications of synapses have been well described in the developing visual cortex, but the ability for experience to modify synapses in the adult visual cortex is poorly understood. We found that 10 d of late-onset visual deprivation modifies both presynaptic and postsynaptic elements at the layer 4--greater than 2/3 connection in the visual cortex of adult mice, and these changes differ from those observed in juveniles. Although visual deprivation in juvenile mice modifies the subunit composition and increases the current duration of synaptic NMDA receptors (NMDARs), no such effect is observed at synapses between layer 4 and layer 2/3 pyramidal neurons in adult mice. Surprisingly, visual deprivation in adult mice enhances the temporal summation of NMDAR-mediated currents induced by bursts of high-frequency stimulation. The enhanced temporal summation of NMDAR-mediated currents in deprived cortex could not be explained by a reduction in the rate of synaptic depression, because our data indicate that late-onset visual deprivation actually increases the rate of synaptic depression. Biochemical and electrophysiological evidence instead suggest that the enhanced temporal summation in adult mice could be accounted for by a change in the molecular composition of NMDARs at perisynaptic/extrasynaptic sites. Our data demonstrate that the experience-dependent modifications observed in the adult visual cortex are different from those observed during development. These differences may help to explain the unique consequences of sensory deprivation on plasticity in the developing versus mature cortex.
|Anti-Post Synaptic Density Protein 95, clone 6G6-1C9 - Data Sheet|