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Mouse Anti-Rat Syntaxin Monoclonal Antibody, Unconjugated, Clone HPC-1

RRID:AB_477483

Antibody ID

AB_477483

Target Antigen

Syntaxin bovine, rabbit, rat, bovine, rabbit

Proper Citation

(Sigma-Aldrich Cat# S0664, RRID:AB_477483)

Clonality

monoclonal antibody

Comments

Vendor recommendations: Immunohistochemistry, Western Blot, Immunohistochemistry (Frozen sections)

Clone ID

Clone HPC-1

Host Organism

mouse

Laminin β2 Chain Regulates Retinal Progenitor Cell Mitotic Spindle Orientation via Dystroglycan.

  • Serjanov D
  • J. Neurosci.
  • 2018 Jun 27

Literature context:


Abstract:

Vertebrate retinal development follows a pattern during which retinal progenitor cells (RPCs) give rise to all retinal cell types in a highly conserved temporal sequence. RPC proliferation and cell cycle exit are tightly coordinated to ensure proper and timely production of each of the retinal cell types. Extracellular matrix (ECM) plays an important role in eye development, influencing RPC proliferation and differentiation. In this study, we demonstrate that laminins, key ECM components, in the inner limiting membrane, control mitotic spindle orientation by providing environmental cues to the RPCs. In vivo deletion of laminin β2 in mice of both sexes results in a loss RPC basal processes and contact with the ECM, leading to a shift of the mitotic spindle pole orientation toward asymmetric cell divisions. This leads to decreased proliferation and premature RPC pool depletion, resulting in overproduction of rod photoreceptors at the expense of bipolar cells and Müller glia. Moreover, we show that deletion of laminin β2 leads to disruption and mislocalization of its receptors: dystroglycan and β1-integrin. Addition of exogenous β2-containing laminins to laminin β2-deficient retinal explants stabilizes the RPC basal processes and directs their mitotic spindle orientation toward symmetric divisions, leading to increased RPC proliferation, as well as restores proper receptor localization at the retinal surface. Finally, functional blocking of dystroglycan in wild-type retinal explants phenocopies laminin β2 ablation. Our data suggest that dystroglycan-mediated signaling between RPCs and the ECM is of key importance in controlling critical developmental events during retinogenesis.SIGNIFICANCE STATEMENT The mechanisms governing retinogenesis are subject to both intrinsic and extrinsic signaling cues. Although the role of intrinsic signaling has been the subject of many studies, our understanding of the role of the microenvironment in retinal development remains unclear. Using a combination of in vivo and ex vivo approaches, we demonstrate that laminins, key extracellular matrix components, provide signaling cues that control retinal progenitor cell attachment to the basement membrane, mitotic axis, proliferation, and fate adoption. Moreover, we identify, for the first time, dystroglycan as the receptor responsible for directing retinal progenitor cell mitotic spindle orientation. Our data suggest a mechanism where dystroglycan-mediated signaling between the cell and the extracellular matrix controls the proliferative potential of progenitors in the developing CNS.

Funding information:
  • NCI NIH HHS - P01 CA095426(United States)

Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation.

  • Simões AP
  • Cell Death Dis
  • 2018 Feb 20

Literature context:


Abstract:

Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

Funding information:
  • NCRR NIH HHS - 5P20-RR021940(United States)

Differential Expression of NF2 in Neuroepithelial Compartments Is Necessary for Mammalian Eye Development.

  • Moon KH
  • Dev. Cell
  • 2018 Jan 8

Literature context:


Abstract:

The optic neuroepithelial continuum of vertebrate eye develops into three differentially growing compartments: the retina, the ciliary margin (CM), and the retinal pigment epithelium (RPE). Neurofibromin 2 (Nf2) is strongly expressed in slowly expanding RPE and CM compartments, and the loss of mouse Nf2 causes hyperplasia in these compartments, replicating the ocular abnormalities seen in human NF2 patients. The hyperplastic ocular phenotypes were largely suppressed by heterozygous deletion of Yap and Taz, key targets of the Nf2-Hippo signaling pathway. We also found that, in addition to feedback transcriptional regulation of Nf2 by Yap/Taz in the CM, activation of Nf2 expression by Mitf in the RPE and suppression by Sox2 in retinal progenitor cells are necessary for the differential growth of the corresponding cell populations. Together, our findings reveal that Nf2 is a key player that orchestrates the differential growth of optic neuroepithelial compartments during vertebrate eye development.

Funding information:
  • NEI NIH HHS - R01 EY013760()
  • NIAMS NIH HHS - R01 AR050772-09(United States)

The packing density of a supramolecular membrane protein cluster is controlled by cytoplasmic interactions.

  • Merklinger E
  • Elife
  • 2017 Jul 19

Literature context:


Abstract:

Molecule clustering is an important mechanism underlying cellular self-organization. In the cell membrane, a variety of fundamentally different mechanisms drive membrane protein clustering into nanometre-sized assemblies. To date, it is unknown whether this clustering process can be dissected into steps differentially regulated by independent mechanisms. Using clustered syntaxin molecules as an example, we study the influence of a cytoplasmic protein domain on the clustering behaviour. Analysing protein mobility, cluster size and accessibility to myc-epitopes we show that forces acting on the transmembrane segment produce loose clusters, while cytoplasmic protein interactions mediate a tightly packed state. We conclude that the data identify a hierarchy in membrane protein clustering likely being a paradigm for many cellular self-organization processes.

Multiple cell types form the VIP amacrine cell population.

  • Pérez de Sevilla Müller L
  • J. Comp. Neurol.
  • 2017 May 4

Literature context:


Abstract:

Amacrine cells are a heterogeneous group of interneurons that form microcircuits with bipolar, amacrine and ganglion cells to process visual information in the inner retina. This study has characterized the morphology, neurochemistry and major cell types of a VIP-ires-Cre amacrine cell population. VIP-tdTomato and -Confetti (Brainbow2.1) mouse lines were generated by crossing a VIP-ires-Cre line with either a Cre-dependent tdTomato or Brainbow2.1 reporter line. Retinal sections and whole-mounts were evaluated by quantitative, immunohistochemical, and intracellular labeling approaches. The majority of tdTomato and Confetti fluorescent cell bodies were in the inner nuclear layer (INL) and a few cell bodies were in the ganglion cell layer (GCL). Fluorescent processes ramified in strata 1, 3, 4, and 5 of the inner plexiform layer (IPL). All tdTomato fluorescent cells expressed syntaxin 1A and GABA-immunoreactivity indicating they were amacrine cells. The average VIP-tdTomato fluorescent cell density in the INL and GCL was 535 and 24 cells/mm2 , respectively. TdTomato fluorescent cells in the INL and GCL contained VIP-immunoreactivity. The VIP-ires-Cre amacrine cell types were identified in VIP-Brainbow2.1 retinas or by intracellular labeling in VIP-tdTomato retinas. VIP-1 amacrine cells are bistratified, wide-field cells that ramify in strata 1, 4, and 5, VIP-2A and 2B amacrine cells are medium-field cells that mainly ramify in strata 3 and 4, and VIP-3 displaced amacrine cells are medium-field cells that ramify in strata 4 and 5 of the IPL. VIP-ires-Cre amacrine cells form a neuropeptide-expressing cell population with multiple cell types, which are likely to have distinct roles in visual processing.

Efferent innervation of turtle semicircular canal cristae: comparisons with bird and mouse.

  • Jordan PM
  • J. Comp. Neurol.
  • 2015 Jun 1

Literature context:


Abstract:

In the vestibular periphery of nearly every vertebrate, cholinergic vestibular efferent neurons give rise to numerous presynaptic varicosities that target hair cells and afferent processes in the sensory neuroepithelium. Although pharmacological studies have described the postsynaptic actions of vestibular efferent stimulation in several species, characterization of efferent innervation patterns and the relative distribution of efferent varicosities among hair cells and afferents are also integral to understanding how efferent synapses operate. Vestibular efferent markers, however, have not been well characterized in the turtle, one of the animal models used by our laboratory. Here we sought to identify reliable efferent neuronal markers in the vestibular periphery of turtle, to use these markers to understand how efferent synapses are organized, and to compare efferent neuronal labeling patterns in turtle with two other amniotes using some of the same markers. Efferent fibers and varicosities were visualized in the semicircular canal of red-eared turtles (Trachemys scripta elegans), zebra finches (Taeniopygia guttata), and mice (Mus musculus) utilizing fluorescent immunohistochemistry with antibodies against choline acetyltransferase (ChAT). Vestibular hair cells and afferents were counterstained using antibodies to myosin VIIa and calretinin. In all species, ChAT labeled a population of small diameter fibers giving rise to numerous spherical varicosities abutting type II hair cells and afferent processes. That these ChAT-positive varicosities represent presynaptic release sites were demonstrated by colabeling with antibodies against the synaptic vesicle proteins synapsin I, SV2, or syntaxin and the neuropeptide calcitonin gene-related peptide. Comparisons of efferent innervation patterns among the three species are discussed.

Müller cells express the cannabinoid CB2 receptor in the vervet monkey retina.

  • Bouskila J
  • J. Comp. Neurol.
  • 2013 Aug 1

Literature context:


Abstract:

The presence of the cannabinoid receptor type 1 (CB1R) has been largely documented in the rodent and primate retinae in recent years. There is, however, some controversy concerning the presence of the CB2 receptor (CB2R) within the central nervous system. Only recently, CB2R has been found in the rodent retina, but its presence in the primate retina has not yet been demonstrated. The aim of this study was twofold: 1) to characterize the distribution patterns of CB2R in the monkey retina and compare this distribution with that previously reported for CB1R and 2) to resolve the controversy on the presence of CB2R in the neural component of the retina. We therefore thoroughly examined the cellular localization of CB2R in the vervet monkey (Chlorocebus sabeus) retina, using confocal microscopy. Our results demonstrate that CB2R, like CB1R, is present throughout the retinal layers, but with striking dissimilarities. Double labeling of CB2R and glutamine synthetase shows that CB2R is restricted to Müller cell processes, extending from the internal limiting membrane, with very low staining, to the external limiting membrane, with heavy labeling. We conclude that CB2R is indeed present in the retina but exclusively in the retinal glia, whereas CB1R is expressed only in the neuroretina. These results extend our knowledge on the expression and distribution of cannabinoid receptors in the monkey retina, although further experiments are still needed to clarify their role in retinal functions.

Funding information:
  • NIGMS NIH HHS - GM081084(United States)

Cannabinoid receptor type 1 expression during postnatal development of the rat retina.

  • Zabouri N
  • J. Comp. Neurol.
  • 2011 May 1

Literature context:


Abstract:

Cannabinoid receptor type 1 (CB1R) participates in developmental processes in the central nervous system (CNS). The rodent retina represents an interesting and valuable model for studying CNS development, because it contains well-identified cell types with clearly established and distinct developmental timelines. Very little is known about the distribution or function of CB1R in the developing retina. In this study, we investigated the expression pattern of CB1R in the rat retina during all stages of postnatal development. Western blots were performed on retinal tissue at different time points between P1 and adulthood. In order to identify the cells expressing the receptor and the age at which this expression started, immunohistochemical co-staining was carried out for CB1R and markers of the different cell types comprising the retina. CB1R was already present at P1 in various cell types, i.e., ganglion, amacrine, horizontal, and mitotic cells. In the course of development, it appeared in cone photoreceptors and bipolar cells. For some cell types (bipolar, Müller, and some amacrine cells), CB1R was transiently expressed, suggesting a potential role of this receptor in developmental processes, such as migration, morphological changes, sub-identity acquisition, and patterned retinal spontaneous activity. Our results also indicated that CB1R is largely expressed in the adult retina (cone photoreceptors and horizontal, most amacrine, and retinal ganglion cells), and may therefore contribute to retinal functions. Overall these results indicate that, as shown in other structures of the brain, CB1R could play an instrumental role in the development and function of the retina.

Funding information:
  • NIMH NIH HHS - R01 MH076136(United States)

Localization of Kv1.3 channels in presynaptic terminals of brainstem auditory neurons.

  • Gazula VR
  • J. Comp. Neurol.
  • 2010 Aug 15

Literature context:


Abstract:

Elimination of the Kv1.3 voltage-dependent potassium channel gene produces striking changes in the function of the olfactory bulb, raising the possibility that this channel also influences other sensory systems. We have examined the cellular and subcellular localization of Kv1.3 in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, a nucleus in which neurons fire at high rates with high temporal precision. A clear gradient of Kv1.3 immunostaining along the lateral to medial tonotopic axis of the MNTB was detected. Highest levels were found in the lateral region of the MNTB, which corresponds to neurons that respond selectively to low-frequency auditory stimuli. Previous studies have demonstrated that MNTB neurons and their afferent inputs from the cochlear nucleus express three other members of the Kv1 family, Kv1.1, Kv1.2, and Kv1.6. Nevertheless, confocal microscopy of MNTB sections coimmunostained for Kv1.3 with these subunits revealed that the distribution of Kv1.3 differed significantly from other Kv1 family subunits. In particular, no axonal staining of Kv1.3 was detected, and most prominent labeling was in structures surrounding the somata of the principal neurons, suggesting specific localization to the large calyx of Held presynaptic endings that envelop the principal cells. The presence of Kv1.3 in presynaptic terminals was confirmed by coimmunolocalization with the synaptic markers synaptophysin, syntaxin, and synaptotagmin and by immunogold electron microscopy. Kv1.3 immunogold particles in the terminals were arrayed along the plasma membrane and on internal vesicular structures. To confirm these patterns of staining, we carried out immunolabeling on sections from Kv1.3(-/-) mice. No immunoreactivity could be detected in Kv1.3(-/-) mice either at the light level or in immunogold experiments. The finding of a tonotopic gradient in presynaptic terminals suggests that Kv1.3 may regulate neurotransmitter release differentially in neurons that respond to different frequencies of sound.

Funding information:
  • NIBIB NIH HHS - R01 EB002010(United States)

Synaptic activity-related classical protein kinase C isoform localization in the adult rat neuromuscular synapse.

  • Besalduch N
  • J. Comp. Neurol.
  • 2010 Jan 10

Literature context:


Abstract:

Protein kinase C (PKC) is essential for signal transduction in a variety of cells, including neurons and myocytes, and is involved in both acetylcholine release and muscle fiber contraction. Here, we demonstrate that the increases in synaptic activity by nerve stimulation couple PKC to transmitter release in the rat neuromuscular junction and increase the level of alpha, betaI, and betaII isoforms in the membrane when muscle contraction follows the stimulation. The phosphorylation activity of these classical PKCs also increases. It seems that the muscle has to contract in order to maintain or increase classical PKCs in the membrane. We use immunohistochemistry to show that PKCalpha and PKCbetaI were located in the nerve terminals, whereas PKCalpha and PKCbetaII were located in the postsynaptic and the Schwann cells. Stimulation and contraction do not change these cellular distributions, but our results show that the localization of classical PKC isoforms in the membrane is affected by synaptic activity.

Funding information:
  • NIAMS NIH HHS - R37 AR038648-21(United States)

Localization of Kv2.2 protein in Xenopus laevis embryos and tadpoles.

  • Gravagna NG
  • J. Comp. Neurol.
  • 2008 Oct 10

Literature context:


Abstract:

Voltage-gated potassium (Kv) channels sculpt neuronal excitability and play important developmental roles. Kv channels consist of pore-forming alpha- and auxiliary subunits. For many Kv alpha-subunits, existing mRNA probes and antibodies have allowed analysis of expression patterns, typically during adult stages. Here, we focus on the Kv2.2 alpha-subunit, for which the mRNA shows broad expression in the embryo and adult. A lack of suitable antibodies, however, has hindered detailed analysis of Kv2.2 protein localization, especially during development. We developed an antibody that specifically recognizes Kv2.2 protein in Xenopus laevis, a vertebrate well suited for study of early developmental stages. The Kv2.2 antibody recognized heterologously expressed Kv2.2 but not the closely related Kv2.1 protein. Immunodetection of the protein showed its presence at St 32 in ventrolateral regions of the hindbrain and spinal cord. At later stages, several sensory tissues (retina, otic, and olfactory epithelia) also expressed Kv2.2 protein. As development progressed in the central nervous system, Kv2.2 protein distribution expanded in close association with the cytoskeletal marker alpha-tubulin, consistent with growth of neuronal tracts. We analyzed the subcellular distribution of Kv2.2 protein within single cultured neurons. In addition to a surface membrane presence, Kv2.2 protein also resided intracellularly closely associated with alpha-tubulin, as in vivo. Furthermore, in contrast to Kv2.1, Kv2.2 protein localized to long, axonal-like processes, consistent with its in vivo location in tracts. Despite their primary sequence similarity, the contrasting localizations of Kv2.1 and Kv2.2 support different roles for the two during development and neuronal signaling.

Anatomical and neurochemical characterization of dopaminergic interplexiform processes in mouse and rat retinas.

  • Witkovsky P
  • J. Comp. Neurol.
  • 2008 Sep 10

Literature context:


Abstract:

Dopaminergic (DA) neurons of mouse and rat retinas are of the interplexiform subtype (DA-IPC), i.e., they send processes distally toward the outer retina, exhibiting numerous varicosities along their course. The primary question we addressed was whether distally located DA-IPC varicosities, identified by tyrosine hydroxylase (TH) immunoreactivity, had the characteristic presynaptic proteins associated with calcium-dependent vesicular release of neurotransmitter. We found that TH immunoreactive varicosities in the outer retina possessed vesicular monoamine transporter 2 and vesicular GABA transporter, but they lacked immunostaining for any of nine subtypes of voltage-dependent calcium channel. Immunoreactivity for other channels that may permit calcium influx such as certain ionotropic glutamate receptors and canonical transient receptor potential channels (TRPCs) was similarly absent, although DA-IPC varicosities did show ryanodine receptor immunoreactivity, indicating the presence of intracellular calcium stores. The synaptic vesicle proteins sv2a and sv2b and certain other proteins associated with the presynaptic membrane were absent from DA-IPC varicosities, but the vesicular SNARE protein, vamp2, was present in a fraction of those varicosities. We identified a presumed second class of IPC that is GABAergic but not dopaminergic. Outer retinal varicosities of this putative GABAergic IPC did colocalize synaptic vesicle protein 2a, suggesting they possessed a conventional vesicular release mechanism.

Sox9 is expressed in mouse multipotent retinal progenitor cells and functions in Müller glial cell development.

  • Poché RA
  • J. Comp. Neurol.
  • 2008 Sep 20

Literature context:


Abstract:

It is widely accepted that the process of retinal cell fate determination is under tight transcriptional control mediated by a combinatorial code of transcription factors. However, the exact repertoire of factors necessary for the genesis of each retinal cell type remains to be fully defined. Here we show that the HMG-box transcription factor, Sox9, is expressed in multipotent mouse retinal progenitor cells throughout retinogenesis. We also find that Sox9 is downregulated in differentiating neuronal populations, yet expression in Müller glial cells persists into adulthood. Furthermore, by employing a conditional knockout approach, we show that Sox9 is essential for the differentiation and/or survival of postnatal Müller glial cells.

Immunocytochemical analysis of syntaxin-1 in rat circumvallate taste buds.

  • Yang R
  • J. Comp. Neurol.
  • 2007 Jun 20

Literature context:


Abstract:

Mammalian buds contain a variety of morphological taste cell types, but the type III taste cell is the only cell type that has synapses onto nerve processes. We hypothesize that taste cell synapses utilize the SNARE protein machinery syntaxin, SNAP-25, and synaptobrevin, as is used by synapses in the central nervous system (CNS) for Ca2+-dependent exocytosis. Previous studies have shown that taste cells with synapses display SNAP-25- and synaptobrevin-2-like immunoreactivity (LIR) (Yang et al. [2000a] J Comp Neurol 424:205-215, [2004] J Comp Neurol 471:59-71). In the present study we investigated the presynaptic membrane protein, syntaxin-1, in circumvallate taste buds of the rat. Our results indicate that diffuse cytoplasmic and punctate syntaxin-1-LIR are present in different subsets of taste cells. Diffuse, cytoplasmic syntaxin-1-LIR is present in type III cells while punctate syntaxin-1-LIR is present in type II cells. The punctate syntaxin-1-LIR is believed to be associated with Golgi bodies. All of the synapses associated with syntaxin-1-LIR taste cells are from type III cells onto nerve processes. These results support the proposition that taste cell synapses use classical SNARE machinery such as syntaxin-1 for neurotransmitter release in rat circumvallate taste buds.

Funding information:
  • European Research Council - 293549(International)

Dynamic expression of the basic helix-loop-helix transcription factor neuroD in the rod and cone photoreceptor lineages in the retina of the embryonic and larval zebrafish.

  • Ochocinska MJ
  • J. Comp. Neurol.
  • 2007 Mar 1

Literature context:


Abstract:

NeuroD is a basic helix-loop-helix (bHLH) transcription factor critical for determining neuronal cell fate and regulating withdrawal from the cell cycle. We showed previously that, in goldfish, neuroD is expressed in the rod photoreceptor lineage, and we inferred that neuroD is also expressed in a subset of amacrine cells and nascent cone photoreceptors. Here we extended that study by examining the temporal and spatial expression pattern of neuroD in the embryonic and larval zebrafish and by identifying the cell types that express this gene. NeuroD expression in the developing zebrafish retina is dynamic, spanning early retinogenesis and the maturation of cone photoreceptors. In early retinogenesis neuroD expression expands from a small patch in the ventronasal retina, through the remaining retinal neuroepithelium. As retinogenesis progresses, neuroD expression becomes restricted to amacrine cells, immature cones, and cells of rod and cone lineages. This expression achieves an adult pattern by 96 hours postfertilization (hpf), whereupon the temporal pattern of neuroD expression in central retina is spatially recapitulated at the germinative margin. The cellular pattern of expression suggests that neuroD regulates aspects of rod and cone genesis, but through separate cellular lineages. Furthermore, neuroD is coexpressed with the cone-rod-homeobox transcription factor (Crx) in putative cone progenitors and nascent cone photoreceptors, suggesting that, in the zebrafish retina, as in other vertebrate retinas, similar genetic cascades regulate photoreceptor genesis and maturation.

Funding information:
  • NIA NIH HHS - F31 AG044061(United States)