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VGlut1 neurotransmitter transporter antibody

RRID:AB_2187693

Antibody ID

AB_2187693

Target Antigen

VGlut1 neurotransmitter transporter null

Proper Citation

(UC Davis/NIH NeuroMab Facility Cat# 75-066, RRID:AB_2187693)

Clonality

monoclonal antibody

Comments

Originating manufacturer of this product. Applications: IB, ICC, IHC, WB. Validation status: IF or IB (Pass), IB in brain (Pass), IHC in brain (Pass), KO (ND).

Clone ID

N28/9

Host Organism

mouse

Rescue of Learning and Memory Deficits in the Human Nonsyndromic Intellectual Disability Cereblon Knock-Out Mouse Model by Targeting the AMP-Activated Protein Kinase-mTORC1 Translational Pathway.

  • Bavley CC
  • J. Neurosci.
  • 2018 Mar 14

Literature context:


Abstract:

A homozygous nonsense mutation in the cereblon (CRBN) gene results in autosomal recessive, nonsyndromic intellectual disability that is devoid of other phenotypic features, suggesting a critical role of CRBN in mediating learning and memory. In this study, we demonstrate that adult male Crbn knock-out (CrbnKO) mice exhibit deficits in hippocampal-dependent learning and memory tasks that are recapitulated by focal knock-out of Crbn in the adult dorsal hippocampus, with no changes in social or repetitive behavior. Cellular studies identify deficits in long-term potentiation at Schaffer collateral CA1 synapses. We further show that Crbn is robustly expressed in the mouse hippocampus and CrbnKO mice exhibit hyperphosphorylated levels of AMPKα (Thr172). Examination of processes downstream of AMP-activated protein kinase (AMPK) finds that CrbnKO mice have a selective impairment in mediators of the mTORC1 translation initiation pathway in parallel with lower protein levels of postsynaptic density glutamatergic proteins and higher levels of excitatory presynaptic markers in the hippocampus with no change in markers of the unfolded protein response or autophagy pathways. Acute pharmacological inhibition of AMPK activity in adult CrbnKO mice rescues learning and memory deficits and normalizes hippocampal mTORC1 activity and postsynaptic glutamatergic proteins without altering excitatory presynaptic markers. Thus, this study identifies that loss of Crbn results in learning, memory, and synaptic defects as a consequence of exaggerated AMPK activity, inhibition of mTORC1 signaling, and decreased glutamatergic synaptic proteins. Thus, CrbnKO mice serve as an ideal model of intellectual disability to further explore molecular mechanisms of learning and memory.SIGNIFICANCE STATEMENT Intellectual disability (ID) is one of the most common neurodevelopmental disorders. The cereblon (CRBN) gene has been linked to autosomal recessive, nonsyndromic ID, characterized by an intelligence quotient between 50 and 70 but devoid of other phenotypic features, making cereblon an ideal protein for the study of the fundamental aspects of learning and memory. Here, using the cereblon knock-out mouse model, we show that cereblon deficiency disrupts learning, memory, and synaptic function via AMP-activated protein kinase hyperactivity, downregulation of mTORC1, and dysregulation of excitatory synapses, with no changes in social or repetitive behaviors, consistent with findings in the human population. This establishes the cereblon knock-out mouse as a model of pure ID without the confounding behavioral phenotypes associated with other current models of ID.

Funding information:
  • Medical Research Council - MC_U137761446(United Kingdom)
  • NIDA NIH HHS - R01 DA029122()

Pharmacological Rescue of Long-Term Potentiation in Alzheimer Diseased Synapses.

  • Prieto GA
  • J. Neurosci.
  • 2017 Feb 1

Literature context:


Abstract:

Long-term potentiation (LTP) is an activity-dependent and persistent increase in synaptic transmission. Currently available techniques to measure LTP are time-intensive and require highly specialized expertise and equipment, and thus are not well suited for screening of multiple candidate treatments, even in animal models. To expand and facilitate the analysis of LTP, here we use a flow cytometry-based method to track chemically induced LTP by detecting surface AMPA receptors in isolated synaptosomes: fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). First, we demonstrate that FASS-LTP is simple, sensitive, and models electrically induced LTP recorded in intact circuitries. Second, we conducted FASS-LTP analysis in two well-characterized Alzheimer's disease (AD) mouse models (3xTg and Tg2576) and, importantly, in cryopreserved human AD brain samples. By profiling hundreds of synaptosomes, our data provide the first direct evidence to support the idea that synapses from AD brain are intrinsically defective in LTP. Third, we used FASS-LTP for drug evaluation in human synaptosomes. Testing a panel of modulators of cAMP and cGMP signaling pathways, FASS-LTP identified vardenafil and Bay-73-6691 (phosphodiesterase-5 and -9 inhibitors, respectively) as potent enhancers of LTP in synaptosomes from AD cases. These results indicate that our approach could provide the basis for protocols to study LTP in both healthy and diseased human brains, a previously unattainable goal. SIGNIFICANCE STATEMENT: Learning and memory depend on the ability of synapses to strengthen in response to activity. Long-term potentiation (LTP) is a rapid and persistent increase in synaptic transmission that is thought to be affected in Alzheimer's disease (AD). However, direct evidence of LTP deficits in human AD brain has been elusive, primarily due to methodological limitations. Here, we analyze LTP in isolated synapses from AD brain using a novel approach that allows testing LTP in cryopreserved brain. Our analysis of hundreds of synapses supports the idea that AD-diseased synapses are intrinsically defective in LTP. Further, we identified pharmacological agents that rescue LTP in AD, thus opening up a new avenue for drug screening and evaluation of strategies for alleviating memory impairments.

Funding information:
  • NIA NIH HHS - P01 AG000538()
  • NIA NIH HHS - P50 AG016573()
  • NIA NIH HHS - R01 AG034667()
  • NIA NIH HHS - R21 AG048506()
  • NINDS NIH HHS - P01 NS045260()

Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts.

  • Loh KH
  • Cell
  • 2016 Aug 25

Literature context:


Abstract:

Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.

Ouabain-induced cochlear nerve degeneration: synaptic loss and plasticity in a mouse model of auditory neuropathy.

  • Yuan Y
  • J. Assoc. Res. Otolaryngol.
  • 2014 Feb 27

Literature context:


Abstract:

Ouabain application to the round window can selectively destroy type-I spiral ganglion cells, producing an animal model of auditory neuropathy. To assess the long-term effects of this deafferentation on synaptic organization in the organ of Corti and cochlear nucleus, and to ask whether surviving cochlear neurons show any post-injury plasticity in the adult, we quantified the peripheral and central synapses of type-I neurons at posttreatment times ranging from 1 to 3 months. Measures of normal DPOAEs and greatly reduced auditory brainstem responses (ABRs) confirmed the neuropathy phenotype. Counts of presynaptic ribbons and postsynaptic glutamate receptor patches in the inner hair cell area decreased with post-exposure time, as did counts of cochlear nerve terminals in the cochlear nucleus. Although these counts provided no evidence of new synapse formation via branching from surviving neurons, the regular appearance of ectopic neurons in the inner hair cell area suggested that neurite extension is not uncommon. Correlations between pathophysiology and histopathology showed that ABR thresholds are very insensitive to even massive neural degeneration, whereas the amplitude of ABR wave 1 is a better metric of synaptic degeneration.

Funding information:
  • NIGMS NIH HHS - GM19351(United States)

Synaptic plasticity in the hippocampus shows resistance to acute ethanol exposure in transgenic mice with astrocyte-targeted enhanced CCL2 expression.

  • Bray JG
  • Neuropharmacology
  • 2013 Apr 1

Literature context:


Abstract:

It has been shown that ethanol exposure can activate astrocytes and microglia resulting in the production of neuroimmune factors, including the chemokine CCL2. The role of these neuroimmune factors in the effects of ethanol on the central nervous system has yet to be elucidated. To address this question, we investigated the effects of ethanol on synaptic transmission and plasticity in the hippocampus from mice that express elevated levels of CCL2 in the brain and their non-transgenic littermate controls. The brains of the transgenic mice simulate one aspect of the alcoholic brain, chronically increased levels of CCL2. We used extracellular field potential recordings in acutely isolated hippocampal slices to identify neuroadaptive changes produced by elevated levels of CCL2 and how these neuroadaptive changes affect the actions of acute ethanol. Results showed that synaptic transmission and the effects of ethanol on synaptic transmission were similar in the CCL2-transgenic and non-transgenic hippocampus. However, long-term potentiation (LTP), a cellular mechanism thought to underlie learning and memory, in the CCL2-transgenic hippocampus was resistant to the ethanol-induced depression of LTP observed in the non-transgenic hippocampus. Consistent with these results, ethanol pretreatment significantly impaired cued and contextual fear conditioning in non-transgenic mice, but had no effect in CCL2-transgenic mice. These data show that chronically elevated levels of CCL2 in the hippocampus produce neuroadaptive changes that block the depressing effects of ethanol on hippocampal synaptic plasticity and support the hypothesis that CCL2 may provide a neuroprotective effect against the devastating actions of ethanol on hippocampal function.

Funding information:
  • NIDA NIH HHS - R01 DA016602-10(United States)

Altered synaptic transmission in the hippocampus of transgenic mice with enhanced central nervous systems expression of interleukin-6.

  • Nelson TE
  • Brain Behav. Immun.
  • 2012 Aug 16

Literature context:


Abstract:

Elevated levels of the inflammatory cytokine interleukin-6 (IL-6) occur in a number of CNS disorders. However, little is known about how this condition affects CNS neuronal function. Transgenic mice that express elevated levels of IL-6 in the CNS show cognitive changes, increased propensity for hippocampal seizures and reduced number of inhibitory interneurons, suggesting that elevated levels of IL-6 can cause neuroadaptive changes that alter hippocampal function. To identify these neuroadaptive changes, we measured the levels of protein expression using Western blot analysis and synaptic function using field potential recordings in hippocampus from IL-6 transgenic mice (IL-6 tg) and their non-transgenic (non-tg) littermates. Western blot analysis showed enhanced levels of the GFAP and STAT3 in the IL-6 tg hippocampus compared with the non-tg hippocampus, but no difference for several other proteins. Field potential recordings of synaptic transmission at the Schaffer collateral to CA1 synapse showed enhanced dendritic excitatory postsynaptic potentials and somatic population spikes in the CA1 region of hippocampal slices from IL-6 tg mice compared with slices from non-tg littermate controls. No differences were observed for several forms of short-term and long-term synaptic plasticity between hippocampal slices from IL-6 tg and non-tg mice. These results demonstrate that elevated levels of IL-6 can alter mechanisms involved in the excitability of hippocampal neurons and synapses, an effect consistent with recent evidence indicating that elevated production of IL-6 plays an important role in conditions associated with seizure activity and in other impairments observed in CNS disorders with a neuroinflammatory component.

Funding information:
  • NINDS NIH HHS - NS045195(United States)

Functional and anatomical identification of a vesicular transporter mediating neuronal ATP release.

  • Larsson M
  • Cereb. Cortex
  • 2012 May 18

Literature context:


Abstract:

ATP is known to be coreleased with glutamate at certain central synapses. However, the nature of its release is controversial. Here, we demonstrate that ATP release from cultured rat hippocampal neurons is sensitive to RNAi-mediated knockdown of the recently identified vesicular nucleotide transporter (VNUT or SLC17A9). In the intact brain, light microscopy showed particularly strong VNUT immunoreactivity in the cerebellar cortex, the olfactory bulb, and the hippocampus. Using immunoelectron microscopy, we found VNUT immunoreactivity colocalized with synaptic vesicles in excitatory and inhibitory terminals in the hippocampal formation. Moreover, VNUT immunolabeling, unlike that of the vesicular glutamate transporter VGLUT1, was enriched in preterminal axons and present in postsynaptic dendritic spines. Immunoisolation of synaptic vesicles indicated presence of VNUT in a subset of VGLUT1-containing vesicles. Thus, we conclude that VNUT mediates transport of ATP into synaptic vesicles of hippocampal neurons, thereby conferring a purinergic phenotype to these cells.

Funding information:
  • NIDCR NIH HHS - U01 DE024427(United States)
  • NIMH NIH HHS - MH086928(United States)

Rab3B protein is required for long-term depression of hippocampal inhibitory synapses and for normal reversal learning.

  • Tsetsenis T
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2011 Aug 23

Literature context:


Abstract:

Rab3B, similar to other Rab3 isoforms, is a synaptic vesicle protein that interacts with the Rab3-interacting molecule (RIM) isoforms RIM1α and RIM2α as effector proteins in a GTP-dependent manner. Previous studies showed that at excitatory synapses, Rab3A and RIM1α are essential for presynaptically expressed long-term potentiation (LTP), whereas at inhibitory synapses RIM1α is required for endocannabinoid-dependent long-term depression (referred to as "i-LTD"). However, it remained unknown whether i-LTD also involves a Rab3 isoform and whether i-LTD, similar to other forms of long-term plasticity, is important for learning and memory. Here we show that Rab3B is highly enriched in inhibitory synapses in the CA1 region of the hippocampus. Using electrophysiological recordings in acute slices, we demonstrate that knockout (KO) of Rab3B does not alter the strength or short-term plasticity of excitatory or inhibitory synapses but does impair i-LTD significantly without changing classical NMDA receptor-dependent LTP. Behaviorally, we found that Rab3B KO mice exhibit no detectable changes in all basic parameters tested, including the initial phase of learning and memory. However, Rab3B KO mice did display a selective enhancement in reversal learning, as measured using Morris water-maze and fear-conditioning assays. Our data support the notion that presynaptic forms of long-term plasticity at excitatory and inhibitory synapses generally are mediated by a common Rab3/RIM-dependent pathway, with various types of synapses using distinct Rab3 isoforms. Moreover, our results suggest that i-LTD contributes to learning and memory, presumably by stabilizing circuits established in previous learning processes.

Funding information:
  • NCATS NIH HHS - UL1 TR000004(United States)

Wingless-type family member 5A (Wnt-5a) stimulates synaptic differentiation and function of glutamatergic synapses.

  • Varela-Nallar L
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2010 Dec 7

Literature context:


Abstract:

Growing evidence indicates that Wingless-type (Wnt) signaling plays an important role in the maturation of the central nervous system. We report here that Wingless-type family member 5A (Wnt-5a) is expressed early in development and stimulates dendrite spine morphogenesis, inducing de novo formation of spines and increasing the size of the preexisting ones in hippocampal neurons. Wnt-5a increased intracellular calcium concentration in dendritic processes and the amplitude of NMDA spontaneous miniature currents. Acute application of Wnt-5a increased the amplitude of field excitatory postsynaptic potentials (fEPSP) in hippocampal slices, an effect that was prevented by calcium-channel blockers. The physiological relevance of our findings is supported by studies showing that Wnt scavengers decreased spine density, miniature excitatory postsynaptic currents, and fEPSP amplitude. We conclude that Wnt-5a stimulates different aspects of synaptic differentiation and plasticity in the mammalian central nervous system.

Funding information:
  • NHGRI NIH HHS - T32 HG003284(United States)

KCNQ5 reaches synaptic endings in the auditory brainstem at hearing onset and targeting maintenance is activity-dependent.

  • Garcia-Pino E
  • J. Comp. Neurol.
  • 2010 Apr 15

Literature context:


Abstract:

Kv7.5/KCNQ5, a voltage-dependent potassium channel that generates a subthreshold K+ current (also called M-current), is localized in excitatory endings of auditory brainstem nuclei in the adult rat. Here, we focus on how specific targeting develops from birth to adulthood in the rat. We first analyzed by immunocytochemistry the distribution of KCNQ5 during postnatal development of neurons in the anteroventral cochlear nucleus (AVCN) and their targets in the medial nucleus of the trapezoid body (MNTB). From postnatal days (P) 0 to 12, KCNQ5 immunoreactivity was restricted to cell bodies, whereas from P13 onward a shift in labeling pattern was seen, with KCNQ5 immunoreactivity becoming confined to synaptic endings in both the AVCN and MNTB. The developmental synaptic targeting was also accompanied by a downregulation of KCNQ5 transcripts in the cochlear nucleus from P13 onward, as seen with quantitative reverse transcriptase polymerase chain reaction. We further tested whether auditory nerve activity at hearing onset (approximately P12) regulates synaptic targeting of the channel. Cochleae were removed at P10, before hearing onset. In the MNTB, 3 days after cochlear ablation, at P13, KCNQ5 immunoreactivity was seen in calyces of Held, as in normal age-matched controls. However, immunolabeling virtually disappeared from MNTB calyces 40 days after cochlear ablation but reappeared in the somata of neurons in AVCN. These findings suggest that synaptic targeting of KCNQ5 in brainstem auditory neurons occurs around the time of hearing onset, regardless of auditory nerve activity. However, long-term synaptic localization after hearing onset depends on peripheral input.

Funding information:
  • NCRR NIH HHS - RR00165(United States)

Role of the Wnt receptor Frizzled-1 in presynaptic differentiation and function.

  • Varela-Nallar L
  • Neural Dev
  • 2009 Nov 2

Literature context:


Abstract:

BACKGROUND: The Wnt signaling pathway regulates several fundamental developmental processes and recently has been shown to be involved in different aspects of synaptic differentiation and plasticity. Some Wnt signaling components are localized at central synapses, and it is thus possible that this pathway could be activated at the synapse. RESULTS: We examined the distribution of the Wnt receptor Frizzled-1 in cultured hippocampal neurons and determined that this receptor is located at synaptic contacts co-localizing with presynaptic proteins. Frizzled-1 was found in functional synapses detected with FM1-43 staining and in synaptic terminals from adult rat brain. Interestingly, overexpression of Frizzled-1 increased the number of clusters of Bassoon, a component of the active zone, while treatment with the extracellular cysteine-rich domain (CRD) of Frizzled-1 decreased Bassoon clustering, suggesting a role for this receptor in presynaptic differentiation. Consistent with this, treatment with the Frizzled-1 ligand Wnt-3a induced presynaptic protein clustering and increased functional presynaptic recycling sites, and these effects were prevented by co-treatment with the CRD of Frizzled-1. Moreover, in synaptically mature neurons Wnt-3a was able to modulate the kinetics of neurotransmitter release. CONCLUSION: Our results indicate that the activation of the Wnt pathway through Frizzled-1 occurs at the presynaptic level, and suggest that the synaptic effects of the Wnt signaling pathway could be modulated by local activation through synaptic Frizzled receptors.

Funding information:
  • Canadian Institutes of Health Research - (Canada)

An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

  • Linhoff MW
  • Neuron
  • 2009 Mar 12

Literature context:


Abstract:

Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

Distribution of voltage-gated potassium and hyperpolarization-activated channels in sensory afferent fibers in the rat carotid body.

  • Buniel M
  • J. Comp. Neurol.
  • 2008 Oct 1

Literature context:


Abstract:

The chemosensory glomus cells of the carotid body (CB) detect changes in O2 tension. Carotid sinus nerve fibers, which originate from peripheral sensory neurons located within the petrosal ganglion, innervate the CB. Release of transmitter from glomus cells activates the sensory afferent fibers to transmit information to the nucleus of the solitary tract in the brainstem. The ion channels expressed within the sensory nerve terminals play an essential role in the ability of the terminal to initiate action potentials in response to transmitter-evoked depolarization. However, with a few exceptions, the identity of ion channels expressed in these peripheral nerve fibers is unknown. This study addresses the expression of voltage-gated channels in the sensory fibers with a focus on channels that set the resting membrane potential and regulate discharge patterns. By using immunohistochemistry and fluorescence confocal microscopy, potassium channel subunits and HCN (hyperpolarization-activated) family members were localized both in petrosal neurons that expressed tyrosine hydroxylase and in the CSN axons within the carotid body. Channels contributing to resting membrane potential, including HCN2 responsible in part for I(h) current and the KCNQ2 and KCNQ5 subunits thought to underlie the neuronal "M current," were identified in the sensory neurons and their axons innervating the carotid body. In addition, the results presented here demonstrate expression of several potassium channels that shape the action potential and the frequency of discharge, including Kv1.4, Kv1.5, Kv4.3, and K(Ca) (BK). The role of these channels should be considered in interpretation of the fiber discharge in response to perturbation of the carotid body environment.

Rat meningeal and brain microvasculature pericytes co-express the vesicular glutamate transporters 2 and 3.

  • Mathur BN
  • Neurosci. Lett.
  • 2008 Apr 18

Literature context:


Abstract:

Pericytes are small cells that are apposed to brain and meningeal microvasculature and control capillary contraction, thereby regulating local cerebral perfusion. Pericytes respond to exogenously applied glutamate in vitro and express metabotropic glutamate receptors. However, it is unclear if pericytes have the capacity to release glutamate. We therefore determined whether pericytes express vesicular glutamate transporters (VGLUTs), which are considered to be unambiguous markers of cells that use glutamate as an intercellular signaling molecule. Leptomeningeal and brain microvasculature-associated pericytes of the adult rat, as defined by the presence of NG2 proteoglycan, expressed both VGLUT2- and VGLUT3-immunoreactivity, but did not express VGLUT1. Consistent with the hypothesis that pericytes release glutamate, VGLUT2- and VGLUT3-immunoreactivities appeared to be localized to secretory vesicles. These results suggest that glutamate is released from pericytes of the leptomeninges and brain microvasculature, and demonstrate for the first time the co-localization of VGLUT2 and VGLUT3.

Funding information:
  • NINDS NIH HHS - R01 NS085232(United States)