Ultrastructural damage and neuritic beading in cold-stressed spinal neurons with comparisons to NMDA and A23187 toxicity

Emery, Dennis G.; Lucas, Jen Hill

doi:10.1016/0006-8993(95)00726-7

Cited by 38 publications

(35 citation statements)

References 54 publications

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“…Fragmented microtubules and loss of microtubule-associated proteins, such as MAP2, have been found in dendrites after ischemic injury (Yamamoto et al, 1986;Tomimoto and Yanagihara, 1992;Emery and Lucas, 1995;Greenwood et al, 2007). We found that microtubule dynamics change with NMDA treatment (Fig.…”

Section: Discussionmentioning

confidence: 51%

“…Taxol, a microtubule stabilizer, has been shown to reduce dendritic swelling in response to sublethal stimulation of the NMDA receptor (Emery and Lucas, 1995;Furukawa and Mattson, 1995;Park et al, 1996). Taxol was applied to neurons 30 min before NMDA treatment until the end of recording.…”

Section: Organization Of Microtubules Is Disrupted After Nmda Exposurementioning

confidence: 99%

“…It is thought that microtubule integrity relates to varicosity formation and dendrite recovery (Faddis et al, 1997). Indeed, Taxol, the microtubule stabilizer, attenuates glutamate-induced varicosity formation (Emery and Lucas, 1995;Furukawa and Mattson, 1995;Park et al, 1996). In addition, spine retraction and reemergence are associated with modification of cytoskeletal organization.…”

Section: Introductionmentioning

confidence: 99%

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The Role of PSD-95 and Cypin in Morphological Changes in Dendrites Following Sublethal NMDA Exposure

Tseng¹,

Firestein²

2011

J. Neurosci.

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Focal swelling or varicosity formation in dendrites and loss of dendritic spines are the earliest indications of glutamate-induced excitotoxicity. Although it is known that microtubule dynamics play a role in varicosity formation, very little is known about the proteins that directly impact microtubules during focal swelling and dendritic spine loss. Our laboratory has recently reported that the postsynaptic protein PSD-95 and its cytosolic interactor (cypin) regulate the patterning of dendrites in hippocampal neurons. Cypin promotes microtubule assembly, and PSD-95 disrupts microtubule organization. Thus, we hypothesized that cypin and PSD-95 may play a role in altering dendrite morphology and spine number in response to sublethal NMDA-induced excitotoxicity. Using an in vitro model of glutamateinduced toxicity in rat hippocampal cultures, we found that cypin overexpression or PSD-95 knockdown increases the percentage of neurons with varicosities and the number of varicosities along dendrites, decreases the size of varicosities after sublethal NMDA exposure, and protects neurons from NMDA-induced death. In contrast, cypin knockdown or PSD-95 overexpression results in opposite effects. We further show that cypin regulates the density of spines/filopodia: cypin overexpression decreases the number of protrusions per micrometer of dendrite while cypin knockdown results in an opposite effect. Cypin overexpression and PSD-95 knockdown attenuate NMDA-promoted decreases in protrusion density. Thus, we have identified a novel pathway by which the microtubule cytoskeleton is regulated during sublethal changes to dendrites.

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Section: Discussionmentioning

confidence: 51%

Section: Organization Of Microtubules Is Disrupted After Nmda Exposurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Role of PSD-95 and Cypin in Morphological Changes in Dendrites Following Sublethal NMDA Exposure

Tseng¹,

Firestein²

2011

J. Neurosci.

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“…Activation of glutamate receptors results in the formation of varicosities in neuronal processes (28), and the glutamate receptor subunit, GluR1, moves from a cytosolic pool to the plasma membrane during activity in hippocampal neurons (16). These in vitro studies are enlightening because glutamate-evoked morphological changes are implicated in the pathogenesis of ischemic neurotoxicity, and GluR1 redistribution may occur during synaptic plasticity (16).…”

Section: Discussionmentioning

confidence: 99%

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X ₂ -green fluorescent protein receptors

Khakh

Smith²,

Chiu³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X 2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X 2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X 2 receptor distribution on the seconds time scale.ion channel ͉ ATP ͉ filopodia C ationic P2X receptors mediate the ''fast'' milliseconds time scale actions of ATP in the nervous system (1, 2). The identity of most natively expressed P2X receptors is unclear, but many neurons express P2X 2 mRNA, P2X 2 proteins, and functional P2X 2 -like receptors (2). For example, ATP mediates synaptic transmission in a portion of CA1 neurons (3), and postnatal hippocampal neurons express P2X receptors, which include P2X 2 subunits (3-7). Moreover, cytosolic ATP concentration is 1-5 mM, and ATP released during tissue damage activates neuronal P2X receptors in the periphery (1). ATP released as a synaptic transmitter and during ischemia of brain neurons may contribute to pathophysiology, but there are no available data on the cellular consequences of P2X 2 receptor activation or on the dynamic aspects of P2X 2 receptor distribution in brain neurons.This study used P2X 2 receptors tagged with green fluorescent protein (GFP) in a quantitative method to study receptors expressed with recombinant Sindbis virus in embryonic hippocampal neurons. We report (i) the properties of functional GFP-tagged P2X 2 receptors, (ii) an optical and electrophysiological approach to measuring receptor numbers in living cells, and (iii) the cellular effects of P2X receptor activation. Materials and MethodsMolecular Biology. By PCR the P2X 2 stop codon was removed and the FLAG (f) epitope was inserted in frame with the P2X 2 cDNA cDNA (9). In the same PCR we inserted an XhoI site in the DNA. We generated GFP37 (10) with a XhoI site before the start codon and subcloned it into P2X 2 -f between the XhoI site 3Ј of the FLAG epitope and HindIII in the pcDNA3 polylinker to yield P2X 2 -GFP. The P2X 2 -f-GFP fragment was inserted into pSinRep5 between the StuI and ApaI sites, and infective Sindbis particles were generated with the use of the Sindbis Expression System (http:͞͞www.invitrogen.com͞). Site-directed mutagenesis was performed on the cDNAs with the use of synthetic oligonucleotides to generate K69A and T18A mutants (Quick Change; Stratagene).Electrophysiology and Imaging. All cell preparations, twoelectrode voltage-clamp record...

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“…Cell swelling is likely an osmotic consequence of Na + influx through glutamate receptors (Choi, 1987), whereas the ionic dependence of dendritic varicosities is less clear, but may involve Ca2~ions for their initiation or maintenance (Bindokas and Miller, 1995;Faddis et al, 1997). These morphological changes have been associated with neuronal injury both in vitro and in vivo (Olney, 1971;Emery and Lucas, 1995;and references above). Therefore, we examined the relationship of these changes to neuronal Ca2~loading through distinct pathways.…”

Section: Source Specificity Of Ca2~neurotoxicity In Cultured Corticalmentioning

confidence: 99%

Distinct Influx Pathways, Not Calcium Load, Determine Neuronal Vulnerability to Calcium Neurotoxicity

Sattler¹,

Charlton

Hafner

et al. 1998

Journal of Neurochemistry

246

192

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Many forms of neurodegeneration are ascribed to excessive cellular Ca 2~loading (Ca2~hypothesis). We examined quantitatively whether factors other than 1Ca 2l oading were determinants of excitotoxic neurode~'eneration. Cell survival, morphology, free intracellular Cas1 concentration ([Ca2~]), and 45Ca2~accumulation were measured in cultured cortical neurons loaded with known quantities of Ca2through distinct transmembrane pathways triggered by excitatory amino acids, cell membrane depolarization, or Ca2ĩonophores. Contrary to the Ca2h ypothesis, the relationships between Ca2~load and cell survival, free [Ca2~]~, and Ca2~-inducedmorphological alterations depended primarily on the route of Ca2~influx, not the Ca2~load. Notably, Ca2~loading via NMDA receptor channels was toxic, whereas identical Ca2~loads incurred through voltage-sensitive Ca2~channels were completely innocuous. Furthermore, accounting quantitatively for Ca2~loading via NMDA receptors uncovered a previously unreported component of L-glutamate neurotoxicity apparently not mediated by ionotropic or metabotropic glutamate receptors. lt was synergistic with toxicity attributable to glutamate-evoked Ca2~loading, and correlated with enhanced cellular ATP depletion. This previously unrecognized toxic action of glutamate constituted a chief excitotoxic mechanism under conditions producing submaximal Ca2~loading. We conclude that (a) Ca2~neurotoxicity is a function of the Ca2~infltlx pathway, not Ca2 load, and (b) glutamate toxicity may not be restricted to its actions on glutamate receptors.

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Ultrastructural damage and neuritic beading in cold-stressed spinal neurons with comparisons to NMDA and A23187 toxicity

Cited by 38 publications

References 54 publications

The Role of PSD-95 and Cypin in Morphological Changes in Dendrites Following Sublethal NMDA Exposure

The Role of PSD-95 and Cypin in Morphological Changes in Dendrites Following Sublethal NMDA Exposure

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X ₂ -green fluorescent protein receptors

Distinct Influx Pathways, Not Calcium Load, Determine Neuronal Vulnerability to Calcium Neurotoxicity

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Ultrastructural damage and neuritic beading in cold-stressed spinal neurons with comparisons to NMDA and A23187 toxicity

Cited by 38 publications

References 54 publications

The Role of PSD-95 and Cypin in Morphological Changes in Dendrites Following Sublethal NMDA Exposure

The Role of PSD-95 and Cypin in Morphological Changes in Dendrites Following Sublethal NMDA Exposure

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X 2 -green fluorescent protein receptors

Distinct Influx Pathways, Not Calcium Load, Determine Neuronal Vulnerability to Calcium Neurotoxicity

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Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X ₂ -green fluorescent protein receptors