Literature context: nti-GFP VRW Cat No:600-101-215; RRID:AB_218182 Rat polyclonal anti-GFP Nacalai
The neonatal mammal faces an array of sensory stimuli when diverse neuronal types have yet to form sensory maps. How these inputs interact with intrinsic neuronal activity to facilitate circuit assembly is not well understood. By using longitudinal calcium imaging in unanesthetized mouse pups, we show that layer I (LI) interneurons, delineated by co-expression of the 5HT3a serotonin receptor (5HT3aR) and reelin (Re), display spontaneous calcium transients with the highest degree of synchrony among cell types present in the superficial barrel cortex at postnatal day 6 (P6). 5HT3aR Re interneurons are activated by whisker stimulation during this period, and sensory deprivation induces decorrelation of their activity. Moreover, attenuation of thalamic inputs through knockdown of NMDA receptors (NMDARs) in these interneurons results in expansion of whisker responses, aberrant barrel map formation, and deficits in whisker-dependent behavior. These results indicate that recruitment of specific interneuron types during development is critical for adult somatosensory function.
Literature context: ckland, RRID:AB_218182); mouse monoclonal anti-HA (clo
Leukocyte common antigen-receptor protein tyrosine phosphatases (LAR-RPTPs) are hub proteins that organize excitatory and inhibitory synapse development through binding to various extracellular ligands. Here, we report that knockdown (KD) of the LAR-RPTP family member PTPσ reduced excitatory synapse number and transmission in cultured rat hippocampal neurons, whereas KD of PTPδ produced comparable decreases at inhibitory synapses, in both cases without altering expression levels of interacting proteins. An extensive series of rescue experiments revealed that extracellular interactions of PTPσ with Slitrks are important for excitatory synapse development. These experiments further showed that the intracellular D2 domain of PTPσ is required for induction of heterologous synapse formation by Slitrk1 or TrkC, suggesting that interaction of LAR-RPTPs with distinct intracellular presynaptic proteins drives presynaptic machinery assembly. Consistent with this, double-KD of liprin-α2 and -α3 or KD of PTPσ substrates (N-cadherin and p250RhoGAP) in neurons inhibited Slitrk6-induced, PTPσ-mediated heterologous synapse formation activity. We propose a synaptogenesis model in presynaptic neurons involving LAR-RPTP-organized retrograde signaling cascades, in which both extracellular and intracellular mechanisms are critical in orchestrating distinct synapse types.SIGNIFICANCE STATEMENTIn this study, we sought to test the unproven hypothesis that PTPσ and PTPδ are required for excitatory and inhibitory synapse formation/transmission, respectively, in cultured hippocampal neurons, using knockdown-based loss-of-function analyses. We further performed extensive structure-function analyses, focusing on PTPσ-mediated actions, to address the mechanisms of presynaptic assembly at excitatory synaptic sites. Utilizing interdisciplinary approaches, we systematically applied varied set of PTPσ deletion variants, point mutants, and splice variants to demonstrate that both extracellular and intracellular mechanisms are involved in organizing presynaptic assembly. Strikingly, extracellular interactions of PTPσ with heparan sulfates and Slitrks, intracellular interactions of PTPσ with liprin-α and its associated proteins through the D2 domain, as well as distinct substrates are all critical.
Literature context: i-GFP Rockland Cat#600-101-215; RRID:AB_218182 Rabbit polyclonal anti-GFP Life
The spinal cord contains neural networks that enable regionally distinct motor outputs along the body axis. Nevertheless, it remains unclear how segment-specific motor computations are processed because the cardinal interneuron classes that control motor neurons appear uniform at each level of the spinal cord. V2a interneurons are essential to both forelimb and hindlimb movements, and here we identify two major types that emerge during development: type I neurons marked by high Chx10 form recurrent networks with neighboring spinal neurons and type II neurons that downregulate Chx10 and project to supraspinal structures. Types I and II V2a interneurons are arrayed in counter-gradients, and this network activates different patterns of motor output at cervical and lumbar levels. Single-cell RNA sequencing (RNA-seq) revealed type I and II V2a neurons are each comprised of multiple subtypes. Our findings uncover a molecular and anatomical organization of V2a interneurons reminiscent of the orderly way motor neurons are divided into columns and pools.
Literature context: catalog #600-101-21, Rockland (RRID:AB_218182); rabbit anti-CRF, 1:1000, rc70
Corticotropin-releasing factor (CRF) neurons in the hypothalamic paraventricular nucleus (PVN) initiate hypothalamic-pituitary-adrenal axis activity through the release of CRF into the portal system as part of a coordinated neuroendocrine, autonomic, and behavioral response to stress. The recent discovery of neurons expressing CRF receptor type 1 (CRFR1), the primary receptor for CRF, adjacent to CRF neurons within the PVN, suggests that CRF also signals within the hypothalamus to coordinate aspects of the stress response. Here, we characterize the electrophysiological and molecular properties of PVN-CRFR1 neurons and interrogate their monosynaptic connectivity using rabies virus-based tracing and optogenetic circuit mapping in male and female mice. We provide evidence that CRF neurons in the PVN form synapses on neighboring CRFR1 neurons and activate them by releasing CRF. CRFR1 neurons receive the majority of monosynaptic input from within the hypothalamus, mainly from the PVN itself. Locally, CRFR1 neurons make GABAergic synapses on parvocellular and magnocellular cells within the PVN. CRFR1 neurons resident in the PVN also make long-range glutamatergic synapses in autonomic nuclei such as the nucleus of the solitary tract. Selective ablation of PVN-CRFR1 neurons in male mice elevates corticosterone release during a stress response and slows the decrease in circulating corticosterone levels after the cessation of stress. Our experiments provide evidence for a novel intra-PVN neural circuit that is activated by local CRF release and coordinates autonomic and endocrine function during stress responses.SIGNIFICANCE STATEMENT The hypothalamic paraventricular nucleus (PVN) coordinates concomitant changes in autonomic and neuroendocrine function to organize the response to stress. This manuscript maps intra-PVN circuitry that signals via CRF, delineates CRF receptor type 1 neuron synaptic targets both within the PVN and at distal targets, and establishes the role of this microcircuit in regulating hypothalamic-pituitary-adrenal axis activity.
Literature context: anti-GFP Rockland 600-101-215; RRID:AB_218182 Mouse anti-Tubulin, monoclonal
Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and viral transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention.
Literature context: A; Cat#600-101-215, lot # 25297 RRID:AB_218182 2.3 Animals
The corticotropin-releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G-protein coupled receptors, CRF1 and CRF2 . Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF-related peptide, termed PD-sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high-affinity ligand for CRF receptor studies. A radiolabeled analog, [125 I]-PD-sauvagine, with high signal-to-noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor-deficient mice and subtype-specific antagonists, CRF1 and CRF2 binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [125 I]-PD-sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF1 expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF2 in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD-sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.
Literature context: GFP Rockland Cat# 600-101-215; RRID:AB_218182 Anti-Rabbit GABA Sigma Cat# A20
The quiescence of adult neural stem cells (NSCs) is regulated by local parvalbumin (PV) interneurons within the dentate gyrus (DG). Little is known about how local PV interneurons communicate with distal brain regions to regulate NSCs and hippocampal neurogenesis. Here, we identify GABAergic projection neurons from the medial septum (MS) as the major afferents to dentate PV interneurons. Surprisingly, dentate PV interneurons are depolarized by GABA signaling, which is in sharp contrast to most mature neurons hyperpolarized by GABA. Functionally, these long-range GABAergic inputs are necessary and sufficient to maintain adult NSC quiescence and ablating them leads to NSC activation and subsequent depletion of the NSC pool. Taken together, these findings delineate a GABAergic network involving long-range GABAergic projection neurons and local PV interneurons that couples dynamic brain activity to the neurogenic niche in controlling NSC quiescence and hippocampal neurogenesis.
Literature context: vania, USA, 600-101-215, 1:500, RRID:AB_218182), visualized by rabbit anti-goa
The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 s lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.
Literature context: goat anti-GFP (1:500, Rockland, RRID:AB_218182), mouse IgG1 anti-myc, clone 9e
Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.
Literature context: RRID:AB_218182
Spontaneous remyelination occurs after spinal cord injury (SCI), but the extent of myelin repair and identity of the cells responsible remain incompletely understood and contentious. We assessed the cellular origin of new myelin by fate mapping platelet-derived growth factor receptor α (PDGFRα), Olig2+, and P0+ cells following contusion SCI in mice. Oligodendrocyte precursor cells (OPCs; PDGFRα+) produced oligodendrocytes responsible for de novo ensheathment of ∼30% of myelinated spinal axons at injury epicenter 3 months after SCI, demonstrating that these resident cells are a major contributor to oligodendrocyte regeneration. OPCs also produced the majority of myelinating Schwann cells in the injured spinal cord; invasion of peripheral myelinating (P0+) Schwann cells made only a limited contribution. These findings reveal that PDGFRα+ cells perform diverse roles in CNS repair, as multipotential progenitors that generate both classes of myelinating cells. This endogenous repair might be exploited as a therapeutic target for CNS trauma and disease.SIGNIFICANCE STATEMENT Spinal cord injury (SCI) leads to profound functional deficits, though substantial numbers of axons often survive. One possible explanation for these deficits is loss of myelin, creating conduction block at the site of injury. SCI leads to oligodendrocyte death and demyelination, and clinical trials have tested glial transplants to promote myelin repair. However, the degree and duration of myelin loss, and the extent and mechanisms of endogenous repair, have been contentious issues. Here, we use genetic fate mapping to demonstrate that spontaneous myelin repair by endogenous oligodendrocyte precursors is much more robust than previously recognized. These findings are relevant to many types of CNS pathology, raising the possibility that CNS precursors could be manipulated to repair myelin in lieu of glial transplantation.
Literature context: -101-215; RRID:AB_218182 HA (mouse
Membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) bind directly to neuroligin-1 (NL1) and neuroligin-2 (NL2), thereby respectively regulating excitatory and inhibitory synapse development. However, the mechanisms by which MDGAs modulate NL activity to specify development of the two synapse types remain unclear. Here, we determined the crystal structures of human NL2/MDGA1 Ig1-3 complex, revealing their stable 2:2 arrangement with three interaction interfaces. Cell-based assays using structure-guided, site-directed MDGA1 mutants showed that all three contact patches were required for the MDGA's negative regulation of NL2-mediated synaptogenic activity. Furthermore, MDGA1 competed with neurexins for NL2 via its Ig1 domain. The binding affinities of both MDGA1 and MDGA2 for NL1 and NL2 were similar, consistent with the structural prediction of similar binding interfaces. However, MDGA1 selectively associated with NL2, but not NL1, in vivo. These findings collectively provide structural insights into the mechanism by which MDGAs negatively modulate synapse development governed by NLs/neurexins.
Literature context: 0-101215; RRID:AB_218182 Mouse anti
Mature neurons in the adult peripheral nervous system can effectively switch from a dormant state with little axonal growth to robust axon regeneration upon injury. The mechanisms by which injury unlocks mature neurons' intrinsic axonal growth competence are not well understood. Here, we show that peripheral sciatic nerve lesion in adult mice leads to elevated levels of Tet3 and 5-hydroxylmethylcytosine in dorsal root ganglion (DRG) neurons. Functionally, Tet3 is required for robust axon regeneration of DRG neurons and behavioral recovery. Mechanistically, peripheral nerve injury induces DNA demethylation and upregulation of multiple regeneration-associated genes in a Tet3- and thymine DNA glycosylase-dependent fashion in DRG neurons. In addition, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult CNS is attenuated upon Tet1 knockdown. Together, our study suggests an epigenetic barrier that can be removed by active DNA demethylation to permit axon regeneration in the adult mammalian nervous system.
Literature context: -101-215; RRID:AB_218182 Rabbit ant
Postnatal maintenance or regeneration of pancreatic beta cells is considered to occur exclusively via the replication of existing beta cells, but clinically meaningful restoration of human beta cell mass by proliferation has never been achieved. We discovered a population of immature beta cells that is present throughout life and forms from non-beta precursors at a specialized micro-environment or "neogenic niche" at the islet periphery. These cells express insulin, but lack other key beta cell markers, and are transcriptionally immature, incapable of sensing glucose, and unable to support calcium influx. They constitute an intermediate stage in the transdifferentiation of alpha cells to cells that are functionally indistinguishable from conventional beta cells. We thus identified a lifelong source of new beta cells at a specialized site within healthy islets. By comparing co-existing immature and mature beta cells within healthy islets, we stand to learn how to mature insulin-expressing cells into functional beta cells.
Literature context: sitive cells were detected with goat anti-GFP antibody (1/200, Rockland #600-101-215). Astrocytes were detected with
This study was designed to use superparamagnetic iron oxide nanoparticles (SPIONs) as evaluating tools to study monocyte-derived macrophages (MDM)-mediated delivery of small molecular agents into the diseased brains. MDM were tested with different-configured SPIONs at selected concentrations for their impacts on carrier cells' physiological and migratory properties, which were found to depend largely on particle size, coating, and treatment concentrations. SHP30, a SPION of 30-nm core size with oleic acids plus amphiphilic polymer coating, was identified to have high cellular uptake efficiency and cause little cytotoxic effects on MDM. At lower incubation dose (25μg/mL), few alteration was observed in carrier cells' physiological and in vivo migratory functions, as tested in a lipopolysaccharide-induced acute neuroinflammation mouse model. Nevertheless, significant increase in monocyte-to-macrophage differentiation, and decrease in in vivo carrier MDM inflamed-brain homing ability were found in groups treated with a higher dose of SHP30at 100μg/mL. Overall, our results have identified MDM treatment at 25μg/mL SHP30 resulted in little functional changes, provided valuable parameters for using SPIONs as evaluating tools to study MDM-mediated therapeutics carriage and delivery, and supported the concepts of using monocytes-macrophages as cellular vehicles to transport small molecular agents to the brain.
Cholinergic neurons are the major excitatory neurons of the enteric nervous system (ENS), and include intrinsic sensory neurons, interneurons, and excitatory motor neurons. Cholinergic neurons have been detected in the embryonic ENS; however, the development of these neurons has been difficult to study as they are difficult to detect prior to birth using conventional immunohistochemistry. In this study we used ChAT-Cre;R26R-YFP mice to examine the development of cholinergic neurons in the gut of embryonic and postnatal mice. Cholinergic (YFP+) neurons were first detected at embryonic day (E)11.5, and the proportion of cholinergic neurons gradually increased during pre- and postnatal development. At birth, myenteric cholinergic neurons comprised less than half of their adult proportions in the small intestine (25% of myenteric neurons were YFP+ at P0 compared to 62% in adults). The earliest cholinergic neurons appear to mainly project anally. Projections into the presumptive circular muscle were first observed at E14.5. A subpopulation of cholinergic neurons coexpress calbindin through embryonic and postnatal development, but only a small proportion coexpressed neuronal nitric oxide synthase. Our study shows that cholinergic neurons in the ENS develop over a protracted period of time.
In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons.
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.
Secretagogin, a recently cloned member of the EF-hand family of calcium binding proteins, was localized in the mouse, rat, and rabbit retina using immunofluorescence immunohistochemistry. Secretagogin is expressed in subpopulations of ON and OFF cone bipolar cells; however, no immunoreactivity was observed in rod bipolar cells in any of these species. Using subtype-specific markers and mice expressing green fluorescent protein (GFP) within specific cell classes, we found that secretagogin is expressed in Types 2, 3, 4, 5, 6 and possibly Type 8 cone bipolar cells in the mouse retina. The expression pattern in the rat retina differs slightly with expression in cone bipolar cell Types 2, 5, 6, 7, and 8. Evaluation of secretagogin in the developing mouse retina revealed expression as early as postnatal day 6, with OFF cone bipolar cells showing secretagogin expression prior to the ON cone bipolar cells. Secretagogin is a useful marker of cone bipolar cells for studying alterations in bipolar cell morphology during development and degeneration. Further work will be necessary to elucidate the functional role of this protein in bipolar cells.
In addition to its established role in initiating the endocrine arm of the stress response, corticotropin-releasing factor (CRF) can act in the brain to modulate neural pathways that effect coordinated physiological and behavioral adjustments to stress. Although CRF is expressed in a set of interconnected limbic and autonomic cell groups implicated as primary sites of stress-related peptide action, most of these are lacking or impoverished in CRF receptor (CRFR) expression. Understanding the distribution of functional receptor expression has been hindered by the low resolution of ligand binding approaches and the lack of specific antisera, which have supported immunolocalizations at odds with analyses at the mRNA level. We have generated a transgenic mouse that shows expression of the principal, or type 1, CRFR (CRFR1). This mouse expresses GFP in a cellular distribution that largely mimics that of CRFR1 mRNA and is extensively colocalized with it in individual neurons. GFP-labeled cells display indices of activation (Fos induction) in response to central CRF injection. At the cellular level, GFP labeling marks somatic and proximal dendritic morphology with high resolution and is also localized to axonal projections of at least some labeled cell groups. This includes a presence in synaptic inputs to central autonomic structures such as the central amygdalar nucleus, which is implicated as a stress-related site of CRF action, but lacks cellular CRFR1 expression. These findings validate a new tool for pursuing the role of central CRFR signaling in stress adaptation and suggest means by which the pervasive ligand-receptor mismatch in this system may be reconciled.