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I'm looking for resources or any information about the formation of dendritic spines and synaptogenesis, especially in relation to how new connections are formed on a daily basis.
Does the electrotonic signalling along the axons and through the spines cause new connections to be made based on some kind of spatial condition (maybe an electrical or chemical attraction), or is there some larger heuristic here?
Spine formation (spinogenesis) is almost certainly due to chemical, rather than electrical, signalling between neurons. Although there are exceptions (gap junctions, for one), most forms of inter-cellular communication are mediated by chemicals released by one cell and detected by another. You are right that the cues for synaptogenesis are probably localized (the "spatial condition"), but I'm willing to bet the farm that those local cues are chemical in nature.
A recent paper from Kwon and Sabatini (2011) shows that local release of the neurotransmitter glutamate is sufficient to cause a functional spine to form. Glutamate receptors on the dendrite detect the glutamate and a spine forms (within seconds). At least under these conditions, the presynaptic machinery isn't required at all! Of course, in a less reduced preparation, electrical activity in the axon will signal the glutamate release from the presynaptic side. Thus, in this case, spine formation is activity-dependent but is mediated by chemical cues.
Dendritic spines are thought to grow and recede under LTP and LTD, respectively. See (Bosch and Hayoshi 2011) for a review.
From there, much of the synaptogenesis occurs due to surface molecules present both on the dendrite and the presynaptic axon in the growth cone. Localization and guidance are achieved through gradients of growth factors in the developing nervous system See (Kolodkin and Tessier-Lavigne 2011) for a review of all of these mechanics.
How this maps back onto the human CNS and thinking/learning/memorizing is still up for debate, but some of these mechanisms must have been preserved in higher species.
Bosch M, Hayashi Y. (2012) Structural plasticity of dendritic spines. Curr Opin Neurobiol.,22(3):383-8. (Epub 2011 Sep 28).
Kolodkin AL, Tessier-Lavigne M. (2011). Mechanisms and molecules of neuronal wiring: a primer. Cold Spring Harb Perspect Biol., 3(6). [DOI]
Two classes of factors contributing to spinogenesis have been described in the literature, based on whether they can be considered extrinsic or intrinsic to the dendrite (my classification). Here is a short list of evidence in favor of either:
the presence of extracellular glutamate facilitates spine formation in tissue from very young mice (Richards et al., 2005; Kwon and Sabatini, 2011)
new spines preferentially form towards boutons with pre-existing synaptic contacts (Toni et al., 1999, 2007; Knott et al., 2006; Nägerl et al., 2007)
new spines tend to form away from pre-existing spines on the dendrite (Fu et al. 2012)
dendrites with lower spine densities display a higher degree of spinogenesis (Holtmaat et al. 2005)
A proposed mechanism extrinsic to the dendrite that can lead to spinogenesis is glutamate spill-over. Conceivable mechanisms intrinsic to the dendrite that can control spinogenesis can be the competition for resources (structural proteins, mRNA etc).
Dendrites of pyramidal cells in some areas of the cortex and the hippocampus exhibit a relatively high turn-over of spine formation and elimination (Holtmaat et al. 2005, Attardo et al. 2005), at least as compared to axonal boutons (e.g. de Paola 2006). Whereas some of these new spines stabilize, many disappear soon after their formation. This indicates that, at least in part, their creation is not fully specified by the existence of a pre-synaptic partner, if we assume that the pre-synaptic partner continues to "attract" them (and that's a big if). The relative independence of spinogenesis from presynaptic activity is further corroborated by the fact that most new spines lack a synapse (Knott et al., 2006).
It appears, then, that dendrites over-produce spines in order to sample their environment for potential synaptic partners. The advantage of such a mechanism can be seen when considering the potential wiring diagram changes that neurons can achieve with such relatively cost-less microscopic changes (Stepanyants et al. 2002). It is unclear at the moment at what point between spinogenesis and synapse formation presynaptic activity becomes an influencing factor in the intact adult brain. A theory incorporating the existing information on spinogenesis is forthcoming.
Attardo A, Fitzgerald JE, Schnitzer MJ. (2015) Impermanence of dendritic spines in live adult CA1 hippocampus. Nature, 523(7562), 592-596. https://doi.org/10.1038/nature14467
De Paola, V., Holtmaat, A., Knott, G., Song, S., Wilbrecht, L., Caroni, P., & Svoboda, K. (2006). Cell Type-Specific Structural Plasticity of Axonal Branches and Boutons in the Adult Neocortex. Neuron, 49(6), 861-875. https://doi.org/10.1016/j.neuron.2006.02.017
Fu, M., Yu, X., Lu, J., & Zuo, Y. (2012). Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo. Nature, 483(7387), 92-95. https://doi.org/10.1038/nature10844
Holtmaat, A. J. G. D., Trachtenberg, J. T., Wilbrecht, L., Shepherd, G. M., Zhang, X., Knott, G. W., & Svoboda, K. (2005). Transient and Persistent Dendritic Spines in the Neocortex In Vivo. Neuron, 45(2), 279-291. https://doi.org/10.1016/j.neuron.2005.01.003
Knott, G. W., Holtmaat, A., Wilbrecht, L., Welker, E., & Svoboda, K. (2006). Spine growth precedes synapse formation in the adult neocortex in vivo. Nature Neuroscience, 9(9), 1117-1124. https://doi.org/10.1038/nn1747
Kwon, H.-B., & Sabatini, B. L. (2011). Glutamate induces de novo growth of functional spines in developing cortex. Nature, 474(7349), 100-104. https://doi.org/10.1038/nature09986
Nägerl, U. V., Köstinger, G., Anderson, J. C., Martin, K. A. C., & Bonhoeffer, T. (2007). Protracted synaptogenesis after activity-dependent spinogenesis in hippocampal neurons. The Journal of Neuroscience, 27(30), 8149-56. https://doi.org/10.1523/JNEUROSCI.0511-07.2007
Richards, D. A., Mateos, J. M., Hugel, S., de Paola, V., Caroni, P., Gahwiler, B. H., & McKinney, R. A. (2005). Glutamate induces the rapid formation of spine head protrusions in hippocampal slice cultures. Proceedings of the National Academy of Sciences, 102(17), 6166-6171. https://doi.org/10.1073/pnas.0501881102
Stepanyants, A., Hof, P. R., & Chklovskii, D. B. (2002). Geometry and structural plasticity of synaptic connectivity. Neuron, 34(2), 275-88. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11970869
Toni, N., Buchs, P. A., Nikonenko, I., Bron, C. R., & Muller, D. (1999). LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature, 402(6760), 421-5. https://doi.org/10.1038/46574
Toni, N., Teng, E. M., Bushong, E. a, Aimone, J. B., Zhao, C., Consiglio, A.,… Gage, F. H. (2007). Synapse formation on neurons born in the adult hippocampus. Nature Neuroscience, 10(6), 727-734. https://doi.org/10.1038/nn1908
Robust 3D Reconstruction and Identification of Dendritic Spines from Optical Microscopy Imaging
In neuro-biology, the 3D reconstruction of neurons followed by the identification of dendritic spines is essential for studying neuronal morphology, function and biophysical properties. Most existing methods suffer from problems of low reliability, poor accuracy and require much user interaction. In this paper, we present a method to reconstruct dendrites using a surface representation of the neuron. The skeleton of the dendrite is extracted by a procedure based on the medial geodesic function that is robust and topologically correct, and it is used to accurately identify spines. The sensitivity of the algorithm on the various parameters is explored in detail and the method is shown to be robust.
NMDA-Mediated Remodeling of Mature Dendritic Spines and Synapses Is Blocked by Phospho-Mimetic Cofilin S3D .
Our previous studies have shown that activation of the NMDA receptor promotes dendritic spine remodeling in cultured hippocampal neurons (18). To determine whether cofilin underlies this event, we induced NMDA-dependent chemical LTD in cultured hippocampal neurons expressing control GFP, wild-type (WT) cofilin, constitutively active cofilin S3A , or phospho-mimetic cofilin S3D . Control hippocampal neurons exhibited a high occurrence of mature mushroom-shaped dendritic spines with large heads and short necks, resulting in a high spine head area-to-length ratio ( Fig. 1 ). The mature spines were associated with synaptophysin-positive puncta, demonstrating the proximity of presynaptic boutons, and contained the postsynaptic density (PSD) protein PSD-95, as well as the NR2A/B and GluR2 subunits of NMDARs and AMPARs, respectively ( Fig. 1 A𠄿 ). NMDAR activation increased the proportion of immature dendritic spines, indicated by a decrease in spine head size and the spine head area-to-length ratio ( Fig. 1 H and I ), and induced growth of new filopodia resulting in an overall increase in protrusion density ( Fig. 1J ). NMDAR activation also led to a decrease in the numbers of synaptophysin, PSD-95, NR2A/B, and GluR2 puncta that were associated with dendritic spine heads, which could be a result of spine head shrinkage ( Fig. 1 K–N ). There was a subsequent increase in synaptic puncta along the dendritic shaft with no change in the overall number of these pre- and postsynaptic puncta following NMDAR activation, suggesting their redistribution from the spine heads to the dendritic shaft rather than their elimination.
NMDA-mediated remodeling of mature dendritic spines and synapses is blocked by phospho-mimetic cofilin S3D . (A𠄿) Confocal images showing the dendrites of 14 days in vitro (DIV) hippocampal neurons expressing GFP (A, C, and E) or GFP-tagged cofilin S3D (cofilin S3D -GFP B, D, and F). Neurons were untreated (Cntl), or treated with 50 μM NMDA in Mg 2+ -free solution for 5 min to activate NMDA receptors, followed by NMDA washout and 60 min incubation in conditioned media (NMDA). Pre- and postsynaptic sites were identified by immunostaining against synaptophysin (Syn, A and B), PSD-95 (C and D), the NMDAR subunits NR2A/B (A𠄽), and the AMPAR subunit GluR2 (E and F). F-actin was detected by rhodamine-coupled phalloidin (E and F). Arrowheads denote positive puncta that are associated with dendritic spines, and arrows denote positive puncta in the dendritic shaft. (Scale bars, 10 μm.) (G–J) Quantitative analysis of spine length (G), head area (H), head area/length ratio (I), and density (J) in untreated (Cntl) and NMDA-treated (NMDA) neurons expressing GFP, WT cofilin, constitutively active cofilin S3A , or phospho-mimetic cofilin S3D . Histograms show mean ± SEM (n = 100 spines from 7 neurons per condition). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest (*P < 0.05, **P < 0.01, ***P < 0.001). (K–N) Quantitative analysis of the numbers of synaptophysin (Syn-), PSD-95–, NR2A/B-, GluR2-, and Phalloidin (Phall)-positive puncta associated with dendritic spines per 10 μm of dendrite in untreated (Cntl) or NMDA-treated (NMDA) neurons expressing GFP or cofilin S3D . Histograms show mean ± SEM (n = 7 neurons per condition). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest (*P < 0.05, **P < 0.01, ***P < 0.001).
NMDA-induced changes in dendritic spine morphology and the number of synaptic sites associated with dendritic spines were dependent on cofilin activation, as its inhibition by overexpression of phospho-mimetic cofilin S3D blocked NMDA-induced shrinkage of dendritic spine heads and prevented subsequent loss of synaptic proteins from dendritic spines ( Fig. 1 K–N ). In contrast, overexpression of constitutively active cofilin S3A induced dendritic spine head shrinkage, similar to the effects of NMDA treatment. NMDAR activation did not induce further spine head shrinkage in neurons expressing cofilin S3A , perhaps because these spines already exhibit smaller heads before NMDA treatment ( Fig. 1 G–J ). Our results suggest that NMDA may induce remodeling of dendritic spines and synapses through the regulation of cofilin activation in dendritic spines.
NMDAR Activation Promotes Rapid Translocation of Active Cofilin to Dendritic Spines, Which Is Disrupted in β-Arrestin-2 KO Neurons.
We found that in addition to cofilin dephosphorylation, proper localization of active cofilin is essential for its effects on dendritic spines and synapses. Our studies indicate that acute NMDA treatment of 14 days in vitro (DIV) hippocampal neurons results in a rapid translocation of both WT cofilin and the nonphosphorylatable constitutively active cofilin S3A (Movie S1), but not the phospho-mimetic cofilin S3D mutant, to the heads of dendritic spines within 5 min of NMDA treatment ( Fig. 2 ). In addition, cofilin S3A is diffusely distributed throughout the spines and dendritic shaft before NMDA treatment, indicating that dephosphorylation alone is not sufficient to trigger cofilin clustering in dendritic spines.
NMDAR activation promotes rapid translocation of cofilin to dendritic spines, which is disrupted in β-arrestin-2 KO neurons. (A𠄼) Time-lapse fluorescent images showing the dendrites of WT, β-arrestin-1 KO, or β-arrestin-2 KO hippocampal neurons at 14 DIV expressing DsRed (red) and WT cofilin-GFP (A), cofilin S3A -GFP (B), or cofilin S3D -GFP (C), before (Cntl) or 5 min after treatment with NMDA. (Scale bars, 10 μm.) See also Movies S1 and S2. (D–G) Quantitative analysis of the distribution of GFP and GFP-tagged cofilin before and after NMDA treatment is shown by the dendritic spine head-to-base (head/base) ratio of GFP fluorescence, normalized against DsRed fluorescence. A ratio of 1 would indicate uniform distribution of GFP or GFP-tagged cofilin in the spine heads and dendritic shaft (base), whereas a ratio that is significantly ϡ would indicate specific targeting of GFP-tagged cofilin to dendritic spines. Histograms show mean ± SEM (n = 100 spines from five neurons per condition). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest. *P < 0.05, **P < 0.01, ***P < 0.001 compared with respective control a indicates value significantly different from that of untreated WT neurons (P < 0.001) b indicates value significantly different from that of NMDA-treated WT neurons (P < 0.01) c indicates value significantly different from that of NMDA-treated β-arrestin-2 KO neurons (P < 0.001).
The scaffolding proteins β-arrestins, which were recently suggested to regulate cofilin localization in migrating cells (33, 34), were prime candidates for the regulation of cofilin translocation in response to NMDAR activation. First, we examined the effects of NMDA on the localization of WT cofilin, constitutively active cofilin S3A , or inactive phospho-mimetic cofilin S3D in β-arrestin-1 KO and β-arrestin-2 KO hippocampal neurons ( Fig. 2 ). NMDA-triggered translocation of constitutively active cofilin S3A into the spines was seen in WT hippocampal neurons ( Fig. 2 B and F and Movie S1), but was impaired in β-arrestin-2 KO neurons ( Fig. 2 B and F and Movie S2). This reduction in the level of cofilin clustering in spines following NMDA treatment demonstrates that β-arrestin-2 is required for proper translocation of active nonphosphorylatable cofilin S3A to the spines in response to NMDAR activation. In contrast to active nonphosphorylatable cofilin S3A , NMDA-induced translocation of WT cofilin to dendritic spines was not affected in either β-arrestin-1 KO or β-arrestin-2 KO neurons ( Fig. 2 A and E ). It is possible that β-arrestin-1 may regulate cofilin translocation in β-arrestin-2 KO neurons, which is blocked by the substitution of Ser to Ala in active nonphosphorylatable cofilin S3A , whereas β-arrestin-2 controls translocation of active nonphosphorylatable cofilin S3A in β-arrestin-1 KO neurons. These results suggest that both β-arrestins may be involved in the translocation of cofilin, but β-arrestin-2 is necessary for the translocation of the active form of cofilin to the spines in response to NMDAR activation.
β-Arrestin-2 KO Neurons Are Resistant to NMDA-Induced Dendritic Spine Remodeling.
The NMDA-induced translocation of cofilin to spines, which occurred rapidly within 5 min of NMDA treatment, is followed by transformation of mature spines with large heads into immature thin spines with small heads (Fig. S1). To investigate whether the ability of β-arrestin-2 to localize active cofilin into dendritic spines is required for NMDA-induced spine remodeling, we examined the effects of NMDA on spine morphology in hippocampal neurons that lack β-arrestin-1 or β-arrestin-2. Our results indicate that deletion of the scaffolding protein β-arrestin-2 abolished the ability of NMDA to induce dendritic spine remodeling. Whereas NMDA treatment of WT neurons resulted in a more immature spine phenotype, the morphology of β-arrestin-2 KO neurons did not change with NMDA treatment ( Fig. 3 A, B, and F–I ). Statistical analysis showed no significant changes in spine length, spine head area, and the spine head area-to-length ratio in NMDA-treated β-arrestin-2 KO neurons compared with untreated β-arrestin-2 KO neurons ( Fig. 3 F–H ). Although the deletion of β-arrestin-2 blocked NMDA-induced spine remodeling, it did not affect normal development of dendritic spines, as β-arrestin-2 KO neurons developed mature spines with large heads that were similar to WT neurons in 14 DIV hippocampal cultures ( Fig. 3 A, B, and F–J ). In contrast, β-arrestin-1 KO neurons exhibited more immature spines that were longer with smaller heads and lower spine head area-to-length ratios than WT neurons, but phospho-mimetic cofilin S3D rescued a mature spine phenotype in the β-arrestin-1 KO neurons ( Fig. 3 E and J ). This result suggests that β-arrestin-1 is required for normal dendritic spine development and may act to suppress excessive cofilin activity in dendritic spines, resulting in spine maturation, whereas loss of β-arrestin-1 may increase the vulnerability of β-arrestin-1 KO neurons to pathological conditions. Indeed, the overexpression of active nonphosphorylatable cofilin S3A in β-arrestin-1 KO neurons resulted in the formation of structures resembling cofilintin rods ( Fig. 2B ) that have been reported in conditions of cellular stress (35, 36). These results suggest that both the ability of β-arrestin-2 to control NMDA-induced spine remodeling and the β-arrestin-1pendent development of mature spines depend on cofilin activation.
β-Arrestin-2 KO neurons develop normal mature spines that are resistant to spine remodeling induced by NMDA or constitutively active cofilin S3A . (A𠄾) Confocal images showing the dendrites of 14 DIV hippocampal neurons from WT, β-arrestin-1 KO, or β-arrestin-2 KO hippocampal neurons expressing DsRed and GFP (A and B) or DsRed and WT cofilin-GFP (C), cofilin S3A -GFP (D), or cofilin S3D -GFP (E). (B) Neurons were treated with NMDA (50 μM, 5 min), followed by washout and incubation in conditioned media for 60 min. (Scale bar, 10 μm.) (F–K) Quantitative analysis of spine length (F), head area (G), head area/length ratio (H and J), density (I), and percentage of filopodia (K). Graphs display mean ± SEM (n = 600 spines from 12 neurons per condition). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest. *P < 0.05, **P < 0.01, ***P < 0.001 a indicates value significantly different from that of WT neurons expressing GFP, WT cofilin, or cofilin S3D (P < 0.001) b indicates value significantly different from that of β-arrestin-2 KO neurons expressing GFP (P < 0.05), WT cofilin (P < 0.01), or cofilin S3D (P < 0.001) c indicates value significantly different from those of all other conditions of the corresponding genotype (P < 0.001).
β-Arrestin-2 Deletion Affects Dendritic Spine Remodeling Induced by Constitutively Active Cofilin S3A .
If the ability of β-arrestin-2 to regulate the localization of cofilin in dendritic spines underlies NMDA-mediated spine remodeling, then deletion of β-arrestin-2 might also affect dendritic spine remodeling induced by constitutively active cofilin S3A . Indeed, constitutively active cofilin S3A failed to cluster in dendritic spines of β-arrestin-2icient neurons in response to NMDA treatment ( Fig. 2B ), and the ability of constitutively active cofilin S3A to induce dendritic spine remodeling was also disrupted in β-arrestin-2icient neurons ( Fig. 3D ). Whereas constitutively active cofilin S3A induced an immature spine phenotype in WT hippocampal neurons, similar to the effects of NMDA treatment, β-arrestin-2 KO neurons expressing constitutively active cofilin S3A exhibited mature spines with a larger head area-to-length ratio and lower percentage of immature filopodia-like protrusions than either WT neurons expressing constitutively active cofilin S3A or β-arrestin-1 KO neurons ( Fig. 3 D, J, and K ). Our results suggest that β-arrestin-2 plays an important role in the translocation of active cofilin to dendritic spines to regulate spine remodeling in response to NMDA.
Overexpression of β-Arrestin-1 or β-Arrestin-2 Reverses the Effect of β-Arrestin Deletion on Dendritic Spine Morphology Under Normal Synaptic Activity or in Response to NMDA.
We were able to reverse the effects of β-arrestin-1 KO on dendritic spine morphology by overexpressing β-arrestin-1 that was tagged with the FLAG peptide sequence (DYKDDDDK) in β-arrestin-1icient neurons ( Fig. 4 A, C, and E ). Analysis of the distribution of transfected FLAG-tagged β-arrestin-1 revealed diffuse localization throughout both the dendritic shafts and spines ( Fig. 4 C and F ). In addition, overexpression of FLAG-tagged β-arrestin-2 rescued NMDA-induced spine remodeling in β-arrestin-2icient neurons ( Fig. 4 B, D, and E ), confirming that β-arrestin-2 is involved in NMDA-mediated dendritic spine remodeling.
Overexpression of β-arrestin-1 or β-arrestin-2 reverses the effect of β-arrestin deletion on dendritic spine morphology under normal synaptic activity or in response to NMDA. (A𠄽) Confocal images showing the dendrites of 14 DIV hippocampal neurons from β-arrestin-1 KO or β-arrestin-2 KO mice expressing GFP alone (A and B) or GFP with FLAG-tagged β-arrestin-1 (C) or FLAG-tagged β-arrestin-2 (D). (Scale bar, 10 μm.) (E and F) Quantitative analysis of dendritic spine morphology (spine head area/length ratio, E) and the distribution of FLAG-tagged β-arrestins (F). The ratio of β-arrestin immunoreactivity was measured in the spine head vs. the dendritic shaft (spine head/base ratio), in WT or KO neurons expressing FLAG-tagged β-arrestin-1 or -2. FLAG-tagged β-arrestin-1 or -2 was detected by immunostaining against FLAG and levels were normalized to GFP fluorescence. Graphs display mean ± SEM (n = 600 spines from 10 cells per condition). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest (*P < 0.05, ***P < 0.001). (G) Mean binding of GST-tagged β-arrestin-2 was determined for WT cofilin, cofilin S3A , and cofilin S3D . Integrated intensity from replicate experiments (n = 3) was determined and normalized to maximal signal. Curves were fit using a nonlinear regression dose–response model.
We also found that β-arrestin-2 can directly interact with WT cofilin, cofilin S3A , and cofilin S3D , with the highest affinity toward constitutively active cofilin S3A ( Fig. 4G ). These findings are consistent with a model in which β-arrestin-2 binds to activated cofilin and promotes its translocation to dendritic spines. β-Arrestin-2 is also distributed equally among dendritic spines and shafts, but appears to form clusters in response to NMDA treatment, which are detected in the heads of dendritic spines ( Fig. 4 D and F ). These results suggest that both β-arrestins are involved in spine development and maintenance under normal synaptic activity, whereas β-arrestin-2 is also involved in cofilin-mediated dendritic spine remodeling in response to NMDA.
β-Arrestin-2 Mediates NMDA-Induced Cofilin Translocation Independent of Cofilin Dephosphorylation.
β-Arrestins may also regulate cofilin phosphorylation levels through scaffolding cofilin with its kinase LIM kinase 1 (LIMK1, where LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3)-1 or phosphatase chronophin (CIN) (33, 34), so we examined whether NMDA-induced cofilin dephosphorylation is affected in β-arrestin-2 KO neurons. Whereas deletion of β-arrestin-2 affected NMDA-induced translocation of active cofilin to dendritic spines and spine remodeling, it was not sufficient to block NMDA-mediated cofilin dephosphorylation, as we observed reduced levels of phosphorylated cofilin in NMDA-treated β-arrestin-2 KO neurons compared with untreated controls ( Fig. 5 A and B ). Similar to previously published reports, we found that NMDA-induced activation of calcineurin and a Ras-PI3K pathway is responsible for NMDA-mediated cofilin dephosphorylation (37, 38), as concurrent treatment of hippocampal neurons with a calcineurin inhibitor [50 μM cyclosporin A (CycA)] and a PI3K inhibitor [50 μM LY294,002 (LY)] completely blocked NMDA-induced cofilin dephosphorylation in hippocampal neurons ( Fig. 5 A and B ). However, dephosphorylation is not required for cofilin clustering in dendritic spines following NMDAR activation, as inhibition of cofilin dephosphorylation did not prevent NMDA-induced translocation of WT cofilin to dendritic spines, an effect that was blocked by the specific NMDAR inhibitor MK801 ( Fig. 5 C and F ). Nevertheless, inhibition of cofilin dephosphorylation with the calcineurin and PI3K inhibitors did block NMDA-induced changes in dendritic spine morphology, suggesting that cofilin can be translocated to dendritic spines in its phosphorylated form, but fails to remodel spines ( Fig. 5 I–K ). These studies show that both calcineurin- and PI3K-mediated cofilin dephosphorylation and β-arrestin–mediated translocation of cofilin to dendritic spines are necessary for NMDA-mediated dendritic spine remodeling (Fig. S2).
Cofilin dephosphorylation is not required for cofilin translocation to dendritic spines, but is necessary for dendritic spine remodeling. (A and B) Fourteen DIV hippocampal neurons were treated with 50 μM NMDA in Mg 2+ -free solution to activate NMDA receptors for 5 min with or without MK801 (10 μM), cyclosporin A (CycA, 50 μM), or LY294,002 (LY, 50 μM). Cell lysates were subjected to immunoblotting with anti–phospho-cofilin antibodies. The blots were stripped and reprobed against total cofilin. The levels of phospho-cofilin were quantified by densitometry and normalized to total cofilin levels, respectively. Experimental values represent mean ± SEM (n = 5). Statistical analysis was performed using Student's t test (**P < 0.01, ***P < 0.001). (C–E) Time-lapse fluorescent images showing the dendrites of 14 DIV WT hippocampal neurons expressing DsRed (red) and WT cofilin-GFP (C), cofilin S3A -GFP (D), or cofilin S3D -GFP (E), before (Cntl) or 5 min after treatment with NMDA. (Scale bars, 10 μm.) (F–H) Quantitative analysis of the distribution of GFP-tagged cofilin in neurons expressing WT cofilin-GFP (F), cofilin S3A -GFP (G), or cofilin S3D -GFP (H), before and after NMDA treatment, with and without MK801, CycA, or CycA and LY294,002 (CycA+LY). The dendritic spine head-to-base (head/base) ratio of GFP fluorescence is normalized against DsRed fluorescence. Histograms show mean ± SEM (n = 100 spines from 5 neurons per condition). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest. *P < 0.05, ***P < 0.001 compared with respective control a and b indicate values significantly different from those of NMDA-treated neurons without inhibitors (a, P < 0.05 b, P < 0.001) c indicates value significantly different from that of untreated neurons (c, P < 0.01). (I–K) Quantitative analysis of spine length (I), head area (J), and head area/length ratio (K) in WT hippocampal neurons expressing WT cofilin, before (Cntl) and after treatment with NMDA, with and without MK801, CycA, or CycA+LY. Graphs display mean ± SEM (n = 100 spines from 7 cells per condition). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest (*P < 0.05, **P < 0.01, ***P < 0.001).
Deletion of β-Arrestin-1, but Not β-Arrestin-2, Disrupts the Development of Mature Dendritic Spines in the Mouse Hippocampus in Vivo.
We next examined whether development of mature dendritic spines is affected in the hippocampus of β-arrestin-1 KO and β-arrestin-2 KO mice in vivo. CA1 hippocampal neurons from β-arrestin-1 KO mice displayed longer spines with a significantly smaller head area and lower head area-to-length ratio than did either WT or β-arrestin-2 KO neurons ( Fig. 6 A𠄿 and I ). In contrast, β-arrestin-2 KO neurons developed normal mature dendritic spines, similar to those of WT neurons. There were no changes in the number of spines or percentage of filopodia among WT and β-arrestin KO neurons ( Fig. 6 G and H ). Whereas the development of mature dendritic spines is affected in β-arrestin-1 KO neurons, β-arrestin-2 KO neurons develop normal mature spines both in vitro and in vivo, but fail to remodel spines in response to NMDA-dependent chemical LTD in vitro.
Deletion of β-arrestin-1, but not β-arrestin-2, disrupts the development of mature dendritic spines in the mouse hippocampus in vivo. (A𠄼) Confocal images showing the dendrites of CA1 neurons labeled with 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindocarbocyanine perchlorate (DiI) in hippocampal slices from WT, β-arrestin-1 KO, or β-arrestin-2 KO mice. (Scale bar, 10 μm.) (D–I) Quantitative analysis of the spine length (D), head area (E), head area/length ratio (F and I), number of protrusions per 10 μm of dendrite (G), and percentage of filopodia (H). Histograms show mean ± SEM (n = 700 spines from six to eight neurons per condition). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest (**P < 0.01, ***P < 0.001).
β-Arrestin-2 KO Mice Exhibit Normal LTP, but Significantly Impaired LTD, in Acute Hippocampal Slices.
The abnormal NMDA-mediated dendritic spine plasticity observed in the β-arrestin-2 KO neurons in vitro suggested that deletion of β-arrestin-2 might result in altered synaptic plasticity. We induced LTP in acute hippocampal slices with two 1-s duration 100-Hz tetani of Schaffer collaterals (39). A potentiation of 150% of the field excitatory postsynaptic potentials (EPSPs) was found in both WT and β-arrestin-2 KO hippocampi following 60 min of high-frequency stimulation, suggesting that β-arrestin-2 KO mice exhibit normal levels of LTP ( Fig. 7 A and B ).
β-Arrestin-2 KO mice exhibit normal LTP, but significantly impaired LTD, in acute hippocampal slices. (A) LTP of Schaffer collateral-CA1 synapses evoked by two trains of 100-Hz pulses at 1 s duration in WT (solid symbols) and β-arrestin-2 KO (shaded symbols) mice. Graph shows mean ± SEM (WT, n = 7 β-arrestin-2 KO, n = 7). (B) Quantitative analysis of extracellular field excitatory postsynaptic potentials (fEPSPs) for minutes 60 in WT (solid bar) and β-arrestin-2 KO (shaded bar) mice. Histogram shows mean ± SEM Student's t test, not significant (NS). (C) LTD of Schaffer collateral-CA1 synapses evoked by paired-pulse LFS at 1 Hz for 15 min in WT (solid symbols) and β-arrestin-2 KO (shaded symbols) mice. Graph shows mean ± SEM (WT, n = 9 β-arrestin-2 KO, n = 10). (D) Quantitative analysis of fEPSPs for minutes 75 in WT (solid bar) and β-arrestin-2 KO (shaded bar) mice. Histogram shows mean ± SEM Student's t test, *P = 0.027. (E) Input/output (I/O) curves as an indication of basal synaptic transmission for WT (solid symbols) and β-arrestin-2 KO (shaded symbols) mice. Graph shows mean ± SEM (WT, n = 17 β-arrestin-2 KO, n = 15) Student's t test, not significant (NS).
The cultured β-arrestin-2 KO neurons showed resistance to NMDA-induced spine head shrinkage, a mechanism that is associated with LTD, and Rust et al. (22) found significantly altered LTD in conditional cofilin KO mice. Therefore, we next induced LTD in Schaffer collateral-CA1 synapses using paired-pulse low-frequency stimulation (PP-LFS) at 1 Hz for 15 min. Interestingly, LTD was significantly impaired in β-arrestin-2 KO mice, as only a 1.2% depression was detected following 60 min of PP-LFS, compared with a 16.1% depression observed in WT mice ( Fig. 7 C and D ). In addition, EPSPs in the slices from the β-arrestin-2 KO mice returned to baseline 30 min after stimulation, whereas EPSPs in WT slices remained at 85% of baseline 60 min following stimulation. In contrast, there were no differences in input/output (I/O) curves between β-arrestin-2 KO and control mice ( Fig. 7E ), indicating that basal pre- and postsynaptic responses are not altered by knockout of β-arrestin-2. Taken together, these results implicate β-arrestin-2 in cellular mechanisms involving dendritic spine shrinkage and LTD.
β-Arrestin-2icient Mice Display Deficits in Long-Term Spatial Learning.
Resistance to NMDA-induced dendritic spine remodeling and impaired NMDA-dependent LTD suggested that hippocampal-dependent learning and memory may be also affected in the β-arrestin-2 KO mice. To examine the spatial learning abilities of β-arrestin-2 KO mice, we used the radial arm water maze, a highly sensitive test of hippocampal-dependent spatial memory (40, 41). Both WT and β-arrestin-2 KO mice performed similarly during days 1𠄶 of the training, indicating that short-term reference memory was not altered in the β-arrestin-2 KO mice ( Fig. 8 ). However, whereas WT mice continued to improve over the course of 11 d, β-arrestin-2 KO mice failed to show improvements from day 7 to day 11. β-Arrestin-2 KO mice needed significantly more time to locate the hidden platform at the end of the test on days 7 ( Fig. 8 A, C, and E ) and made significantly more errors in the process than did WT mice ( Fig. 8 B, D, and F ). These results reveal a deficit in long-term spatial learning and memory in the β-arrestin-2 KO mice.
β-Arrestin2icient mice display deficits in long-term spatial learning. (A𠄿) Quantitative analysis of the latency to locate the hidden platform (A, C, and E) and number of errors (B, D, and F) in the radial arm water maze over 11 d of testing. Graphs display mean ± SEM (WT, solid symbols, n = 13 β-arrestin-2 KO, shaded symbols, n = 12). (A and B) Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest. a, WT/D11/T4+5 shows significant improvement vs. WT/D1/T4+5 (P < 0.05) b and c, WT/D11/T4+5 shows significant improvement vs. WT/D11/T1 (b, P < 0.001 c, P < 0.05) d, β-arrestin-2 KO/D11/T4+5 is significantly impaired vs. WT/D11/T4+5 (P < 0.05). (B and D) Latency to find the platform (B) and number of errors (C) were averaged for the early days of testing (days 1𠄶) and the late days of testing (days 7) to evaluate long-term performance (**P < 0.01, ***P < 0.001). (E and F) Histograms show latency (E) and number of errors (F) for trials 4 and 5 (averaged). Statistical analysis for each individual day of testing was performed using Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001).
β-Arrestin-2icient Hippocampal Neurons Are Resistant to Aβ-Induced Dendritic Spine and Synapse Loss.
The phospho-mimetic cofilin S3D mutant was shown to be protective in hippocampal slices against spine loss induced by Aβ1 oligomers that are implicated in Alzheimer's disease (AD) (26). In addition, the formation of Aβ-induced cofilintin rods in hippocampal slices and cultured neurons was prevented by enhancing cofilin phosphorylation through modulation of LIMK, slingshot phosphatase (SSH), and CIN (25). Here we have demonstrated that β-arrestin-2icient neurons are resistant to NMDA-induced dendritic spine remodeling due to impaired spatial localization of cofilin in dendritic spines. We therefore tested whether depletion of β-arrestin-2 would also protect neurons against Aβ-mediated spine loss, similar to the effects of phospho-mimetic cofilin S3D , which inhibits endogenous cofilin activity. Whereas Aβ promoted immature spines in WT neurons, β-arrestin-2icient neurons did not display spine remodeling with Aβ treatment ( Fig. 9 A–I ), and β-arrestin-2 deletion was also protective against spine loss induced by Aβ peptide ( Fig. 9J ). In addition, whereas Aβ induced a significant decrease in the overall numbers of synaptophysin-positive presynaptic terminals postsynaptic sites containing PSD-95–, NR2A/B-, and GluR2-positive puncta and phalloidin-positive F-actin clusters in WT hippocampal neurons, β-arrestin-2icient neurons were resistant to these changes induced by Aβ treatment ( Fig. 9 K–M ). The numbers of the pre- and postsynaptic proteins that were associated with dendritic spines also did not change in Aβ-treated β-arrestin-2icient neurons ( Fig. 9 N–P ). These studies suggest that β-arrestin-2 may also be involved in some pathological forms of dendritic spine plasticity, such as Aβ-induced spine loss, and may emerge as a new target for AD therapies.
β-Arrestin-2icient hippocampal neurons are resistant to Aβ-induced dendritic spine and synapse loss. (A𠄿) Confocal images showing GFP-expressing dendrites of 14 DIV hippocampal neurons from WT (A, C, and E) or β-arrestin-2 KO mice (B, D, and F), untreated (Cntl) or with application of 225 pM Aβ1 oligomers for 24 h (Aβ). Pre- and postsynaptic sites were identified by immunostaining against synaptophysin (Syn, A and B), PSD-95 (C and D), the NMDAR subunits NR2A/B (A𠄽), and the AMPAR subunit GluR2 (E and F). F-actin was detected by rhodamine-coupled phalloidin (E and F). Arrowheads denote positive puncta that are associated with dendritic spines. (Scale bars, 10 μm.) (G–J) Quantitative analysis of spine length (G), head area (H), head area/length ratio (I), and density (J) in untreated or Aβ1-treated (Aβ) neurons. Histograms show mean ± SEM (n = 7 neurons per condition). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest (**P < 0.01, ***P < 0.001). (K–P) Quantitative analysis of the total number of synaptophysin (Syn-), PSD-95–, NR2A/B-, GluR2-, and Phalloidin (Phall)-positive puncta detected in both dendritic spines and shafts (K–M) or of the number of puncta associated with dendritic spines (N–P) per 10 μm in Aβ1-treated (Aβ) WT or β-arrestin-2 KO neurons. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple-comparison posttest (*P < 0.05, **P < 0.01, ***P < 0.001).
During early synaptogenesis, dendritic shafts are covered with filopodia, which are long, narrow protrusions that are more transient and contain less F-actin than spines. Neuronal growth cones also display numerous filopodia, but these seem to be somehow distinct. Growth cone filopodia are involved in activity-independent dendritic growth and branching, whereas the filopodia that protrude from the dendrite shaft are involved in activity-dependent synaptogenesis (87). Dendritic filopodia transiently extend and retract from the dendritic shaft with an average lifetime of
10 min (129). Filopodia probably function to maximize the chance encounter between a developing axon and a target dendrite. Once contact is made—either physically via cell-cell adhesions or chemically via locally released signals—a synapse can be initiated and proceed through appropriate maturational steps. Such maturation requires intricate crosstalk between the nascent presynaptic and postsynaptic parts of the synapse (31).
As synapse formation progresses over the course of several days, the numerous dendritic filopodia are gradually replaced by spines (67). It appears that many, but not all, spine synapses result when synapses initially form on filopodia, which then “convert” directly into spines. However, other synapses form directly on the dendritic shaft, followed by the gradual emergence of spines at the site of contact (24). Laboratory observations suggest that both mechanisms can and do occur even within a single neuron (22). It should be noted, however, that not all filopodia are transformed into spines (129). Filopodia are also found on nonspiny neurons during early synaptogenesis (121). Thus multiple factors, including cell-recognition molecules and downstream signals, orchestrate both synapse identity and synapse shape during neuronal maturation.
Dendritic spine abnormalities and cognitive impairment
It has long been recognized that neurodegenerative disease and other neurological pathology often manifests as an alteration in neuronal anatomy and in particular in the number, shape, and distribution of dendritic spines (Goldman-Rakic, 1996 DeFelipe, 2004 Alonso-Nanclares et al., 2005 Glausier and Lewis, 2013 He and Portera-Cailliau, 2013 Licznerski and Duman, 2013 Pozueta et al., 2013 Smith and Villalba, 2013 Villalba and Smith, 2013 Wong and Guo, 2013), most likely correlating with alterations in neuronal signaling and axonal and neuronal death. Additionally, treatments that reduce the cognitive symptoms of neurodegenerative disease also reverse spine pathology (Smith et al., 2009). Unfortunately for patients and clinicians, these alterations can only be observed in small amount of tissues sampled from specific brain areas via biopsy, or post-mortem tissue received from deceased patients. The accumulation of senile plaques and tau bundles are the most well-known anatomical hallmarks of Alzheimer’s disease, but it is synapse loss, exemplified by a reduction in dendritic spine density in the cerebral cortex and hippocampus, that best correlates with disease progression (Moolman et al., 2004). A decrease in length and complexity of the dendritic arbor as well as a significant reduction in spine density in medium spiny neurons of the dopamine receiving areas of the brain have long been observed as pathological hallmarks of Parkinson’s disease (Stephens et al., 2005). Rett syndrome patients display a prominent reduction in dendritic arbor complexity, dendritic spine density, total number of neurons, and total brain volume which appears within the first year of the patient’s life (Belichenko and Dahlström, 1995a,b Armstrong, 2005). That these anatomical abnormalities are recapitulated in animal models (Smrt et al., 2007 Belichenko et al., 2009) of the disease reveals that Rett Syndrome is a disorder of neuronal development and reconfirms the correlation between dendritic spine anatomy and neuronal function or dysfunction.
Epilepsy is a neurological disorder resulting from network hyperactivity in neuronal circuits that causes chronic seizures (Bromfield et al., 2006). The principle hyperactive neurons in epilepsy are found in the medial temporal lobe and are characterized by dense excitatory synaptic inputs through dendritic spines. As one might expect, the chronic hyperactivity found in epilepsy correlates with significant changes in spine density, but it is unclear whether spine loss is a cause of epilepsy or a compensatory change in response to it (Wong and Guo, 2013). Further complicating the issue anatomical, and functional changes in the brain coincide with gene transcriptional chances, but a cause on effect relationship has not been established in either direction (Arion et al., 2006). This situation is not the exception but the rule for post mortem studies of diseased human tissue.
Here animal models of human disease are extremely useful for elucidating the causes and mechanisms of synaptic dysfunction. Not only do they offer the lack of confounding genetic and environmental factors found in human patients, but they offer investigators the potential to study disease progression at defined time points. Still, the use of imperfect animal models of human disease yields an understanding of these diseases and disease mechanisms that is overly simplistic (Chesselet and Carmichael, 2012) and the validity of these models in accurately predicting human disease is highly variable. Only comprehensive integration and iteration of clinical studies and mechanistic studies in animal models (under constant evaluation) can provide an accurate vision of the causes and mechanisms of complex neurodegenerative diseases.
Directed anatomical studies of neurons and populations of neurons along with protein expression and electrophysiological studies have been instrumental in the development of models that predict how the electrical properties of neurons vary with changes in cell shape these works have contributed to a large number of popular software packages that model neuronal activity under various conditions (Hines and Carnevale, 2001 Bower and Beeman, 2007). The field of systems and computational neuroscience is mature and continues to flourish. In addition, a wide range of complementary knowledge can be gained by systematic analysis of available data sets and the acquisition of new high throughput data sets chief among these are image data sets on multiple scales and from a variety of modalities which to data remain largely underutilized.
Under what conditions do dendritic spines form? - Biology
Intersectin-short (intersectin-s) is a multimodule scaffolding protein functioning in constitutive and regulated forms of endocytosis in non-neuronal cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of Drosophila and Caenorhabditis elegans. In vertebrates, alternative splicing generates a second isoform, intersectin-long (intersectin-l), that contains additional modular domains providing a guanine nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is expressed in multiple tissues and cells, including glia, but excluded from neurons, whereas intersectin-l is a neuron-specific isoform. Thus, intersectin-I may regulate multiple forms of endocytosis in mammalian neurons, including SV endocytosis. We now report, however, that intersectin-l is localized to somatodendritic regions of cultured hippocampal neurons, with some juxtanuclear accumulation, but is excluded from synaptophysin-labeled axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV recycling. Instead intersectin-l co-localizes with clathrin heavy chain and adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces the rate of transferrin endocytosis. The protein also co-localizes with F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation during development. Our data indicate that intersectin-l is indeed an important regulator of constitutive endocytosis and neuronal development but that it is not a prominent player in the regulated endocytosis of SVs.
This work was supported by Grants MOP-15396 and MOP-86724 from the Canadian Institutes of Health Research (to P. S. M. and R. A. M.), respectively, and from Natural Sciences and Engineering Rgpin 341942-07 (to R. A. M.).
Supported by a Jeane-Timmins Costello Fellowship from the Montreal Neurological Institute.
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In: Neuroscience , Vol. 122, No. 2, 2003, p. 305-315.
Research output : Contribution to journal › Article › peer-review
T1 - Apolipoprotein E isoform-specific regulation of dendritic spine morphology in apolipoprotein E transgenic mice and Alzheimer's disease patients
N1 - Funding Information: This work was supported by NIH grants AG15408, AG17617 and AG13956.
N2 - Dendritic spines are postsynaptic sites of excitatory input in the mammalian nervous system. Apolipoprotein (apo) E participates in the transport of plasma lipids and in the redistribution of lipids among cells. A role for apoE is implicated in regeneration of synaptic circuitry after neural injury. The apoE4 allele is a major risk factor for late-onset familial and sporadic Alzheimer's disease (AD) and is associated with a poor outcome after brain injury. ApoE isoforms are suggested to have differential effects on neuronal repair mechanisms. In vitro studies have demonstrated the neurotrophic properties of apoE3 on neurite outgrowth. We have investigated the influence of apoE genotype on neuronal cell dendritic spine density in mice and in human postmortem tissue. In order to compare the morphology of neurons developing under different apoE conditions, gene gun labeling studies of dendritic spines of dentate gyrus (DG) granule cells of the hippocampus were carried out in wild-type (WT), human apoE3, human apoE4 expressing transgenic mice and apoE knockout (KO) mice the same dendritic spine parameters were also assessed in human postmortem DG from individuals with and without the apoE4 gene. Quantitative analysis of dendritic spine length, morphology, and number was carried out on these mice at 3 weeks, 1 and 2 years of age. Human apoE3 and WT mice had a higher density of dendritic spines than human E4 and apoE KO mice in the 1 and 2 year age groups (P<0.0001), while at 3 weeks there were no differences between the groups. These age dependent differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of dementia in aged individuals with the apoE4 allele. Significantly in human brain, apoE4 dose correlated inversely with dendritic spine density of DG neurons cell in the hippocampus of both AD (P=0.0008) and aged normal controls (P=0.0015). Our findings provide one potential explanation for the increased cognitive decline seen in aged and AD patients expressing apoE4.
AB - Dendritic spines are postsynaptic sites of excitatory input in the mammalian nervous system. Apolipoprotein (apo) E participates in the transport of plasma lipids and in the redistribution of lipids among cells. A role for apoE is implicated in regeneration of synaptic circuitry after neural injury. The apoE4 allele is a major risk factor for late-onset familial and sporadic Alzheimer's disease (AD) and is associated with a poor outcome after brain injury. ApoE isoforms are suggested to have differential effects on neuronal repair mechanisms. In vitro studies have demonstrated the neurotrophic properties of apoE3 on neurite outgrowth. We have investigated the influence of apoE genotype on neuronal cell dendritic spine density in mice and in human postmortem tissue. In order to compare the morphology of neurons developing under different apoE conditions, gene gun labeling studies of dendritic spines of dentate gyrus (DG) granule cells of the hippocampus were carried out in wild-type (WT), human apoE3, human apoE4 expressing transgenic mice and apoE knockout (KO) mice the same dendritic spine parameters were also assessed in human postmortem DG from individuals with and without the apoE4 gene. Quantitative analysis of dendritic spine length, morphology, and number was carried out on these mice at 3 weeks, 1 and 2 years of age. Human apoE3 and WT mice had a higher density of dendritic spines than human E4 and apoE KO mice in the 1 and 2 year age groups (P<0.0001), while at 3 weeks there were no differences between the groups. These age dependent differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of dementia in aged individuals with the apoE4 allele. Significantly in human brain, apoE4 dose correlated inversely with dendritic spine density of DG neurons cell in the hippocampus of both AD (P=0.0008) and aged normal controls (P=0.0015). Our findings provide one potential explanation for the increased cognitive decline seen in aged and AD patients expressing apoE4.
ASJC Scopus subject areas
In: Journal of Biological Chemistry , Vol. 290, No. 26, 26.06.2015, p. 15909-15920.
Research output : Contribution to journal › Article › peer-review
T1 - Actinin-4 governs dendritic spine dynamics and promotes their remodeling by metabotropic glutamate receptors
N1 - Publisher Copyright: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
N2 - Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca2+-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.
AB - Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca2+-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.
Organotypic slices of the hippocampus cultured for at least 10 days exhibited mature dendritic arbors and dendritic spine populations as imaged by EYFP-based fluorescent reporters introduced biolistically (Fig. (Fig.1 1 A). Three weeks after plating, dissociated neurons exhibited dense spine populations and long dendritic and axonal arbors (Fig. (Fig.1 1 C). These observations are consistent with previously published characterizations of the two hippocampal culture systems (19, 20) and provided a basis for the timing of transfections, pharmacological manipulations, and analyses used in this study.
Examples of the effect of mGluR stimulation on the length of dendritic spines in two hippocampal culture systems. Slices (A and B) and dissociated neurons (C and D) were fixed after treatment with vehicle control (Left) or 100 μM DHPG (Right). Control and treated cultures prepared from the same animals were processed in parallel. Scale bar = 5 μm.
Brief Treatment with DHPG Increases the Average Length of Dendritic Spines.
The average length of spines on the dendrites of dentate gyrus granule cells in hippocampal slice cultures and neurons grown in dissociated culture was increased by brief incubation with the group 1 mGluR agonist DHPG (Fig. (Fig.1). 1 ). Increases in spine length were evident after 30 min of incubation with the mGluR agonist (100 μM), and in some experiments were seen as early as 10 min after treatment. The average radial distance from the spine head to the dendritic shaft increased relative to controls by 35.4 ± 5.7% (n = 10 P < 0.01) after a 30-min incubation with DHPG (Fig. (Fig.2 2 Upper). In dissociated neurons, the effect was similar in magnitude (29.0 ± 5.0% n = 9 P < 0.01 Fig. Fig.2 2 Lower). Cultures in which the mGluR antagonist MCPG was added for 30 min were not different from untreated controls. Prolonged incubation with MCPG (2𠄳 days) did not result in statistically significant changes in spine length overall (Fig. (Fig.2), 2 ), although in some experiments it appeared that there was a trend toward reduced lengths.
The effect of DHPG on the average length of dendritic spines of dentate gyrus granule cells in slice culture and dissociated hippocampal neurons. A 30-min incubation with 100 μM DHPG resulted in a significant increase in the average length of dendritic spines (1.35 ± 0.06 μm) relative to controls acutely treated with AP5˼NQX (0.99 ± 0.03 μm n = 10 experiments, 2𠄳 granule cells per experiment, spines per cell P < 0.001, two-tailed Mann–Whitney test). Similar effects were obtained in dissociated neuronal culture (control 1.03 ± 0.03, DHPG 1.33 ± 0.05 P < 0.001). Prolonged incubation with MCPG did not alter the average length of dendritic spines (dentate granule cells, 0.99 ± 0.03 dissociated neurons, 1.05 ± 0.05).
These DHPG-dependent changes were also obtained when a combination of the NMDA- and AMPA-type ionotropic receptor antagonists AP5 and CNQX was included in the media (data not shown). Spine lengths in cultures treated with AP5˼NQX in the absence of DHPG were not different from those in non-treated controls. These results argue for a mechanism that requires direct activation of postsynaptic group 1 metabotropic receptors, rather than an indirect effect through postsynaptic ionotropic receptors triggered by glutamate release following DHPG binding to presynaptic receptors (21).
DHPG Shifts the Distribution of Spine Lengths with Minimal Changes in Spine Density.
Changes in the length of dendritic spines induced by mGluR stimulation were also reflected in frequency distribution analyses of spine length and type. Fig. Fig.3 3 shows the relative frequencies of dendritic spines grouped into 0.3-μm bins. The distribution of spine lengths was skewed toward longer spines with DHPG treatment for both dentate granule cells (Fig. (Fig.3 3 A) and dissociated neurons (Fig. (Fig.3 3 B). However, measurements of spine density were slightly different between control (70.9 ± 4.1 spines per 50 μm, n = 7) and DHPG-treated cultures (82.6 ± 5.1 spines per 50 μm, n = 9 P = 0.071 Mann–Whitney U test, two-tailed). The quantitative results suggest that the observed changes result mainly from the growth of existing spines.
Frequency distribution histograms of dendritic spine length in control and DHPG-treated cultures. (Upper) The length of spines on the dendrites of dentate granule cells in slice culture were measured and segregated into bins of 0.3 μm. The composite frequency distribution from 10 experiments shows that the occurrence of longer spines increases whereas that of shorter spines decreases. (Lower) The same effect is evident in composite frequency distributions of spine lengths in dissociated neuronal cultures.
An analysis of spine types, based on a classification scheme that takes into account overall length and spine head and neck shape characteristics (see Methods) revealed a greater proportion of spines resembling filapodial extensions and of non-“mushroom” type spines. In Fig. Fig.4, 4 , examples of four spine types discriminated by using this classification scheme are labeled throughout an image of a DHPG-treated dendrite. In the Lower panel, analysis of the spine types shows that the greatest effect of DHPG is on the number of thin spines (type 3) and on those resembling filapodial extensions (type 4) a slight decrease in the proportion of type 1 spines seems to accompany these effects. The relative abundance of the classical mushroom-shaped spines (type 2) was similar to that in cultures treated acutely with AP5 + CNQX or with MCPG for 2𠄳 days.
Example of spine types and analysis of shifts in the relative abundance of these types accompanying DHPG treatment. (A) Maximum intensity projection of an image stack taken from a DHPG-treated dentate granule cell. Four spine types are labeled, classified as described in Methods and differentiated with respect to length and shape characteristics. (B) Relative abundance of spine types in control slices and in slices treated acutely with DHPG or chronically for 2𠄳 days with MCPG (control and DHPG treatments also included AP5 + CNQX). Spines identified as type 3 or 4, characterized by longer, thinner profiles with smaller spine heads, are increased in frequency with DHPG treatment whereas shorter (“nubbin”) spines, type 1, are decreased. A slight shift in the opposite direction with chronic MCPG is seen in this experiment, but this result did not occur reliably enough to influence the average spine length. The proportion of type 2 spines, the classic mushroom-shaped profiles, did not change with DHPG treatment.
Perturbation of Calcium Mobilization and of mRNA Translation Inhibits the Effects of DHPG.
Attenuation of calcium mobilization and protein synthesis diminished the effects of DHPG on spine length. Chelation of internal calcium by treating slice cultures with BAPTA blocked the DHPG-induced increases in spine length (DHPG 1.29 ± 0.04 μm DHPG + chelator 0.98 ± 0.05 μm Controls 1.00 ± 0.04 μm). Inhibition of protein synthesis by preincubation of slices with puromycin (100 μM) for 30 min resulted in a slight decrease in average spine length following subsequent coincubation with DHPG (Fig. (Fig.5 5 A). Treatment with puromycin alone did not change the average length of dendritic spines on dentate granule cell dendrites.
(A) The effect of puromycin on DHPG-induced spine lengthening in hippocampal slice cultures. Mean spine lengths in slices treated with puromycin (Puro), DHPG, and DHPG in the presence of puromycin (DHPG + Puro) are graphed with sample images from each treatment above. (B) The effect of simultaneous manipulation of group 1 mGluRs and protein synthesis on spine density. The average number of dendritic spines per 50-μm segment of secondary dendrites of dentate granule cells was not significantly different from controls (70.9 ± 4.1, n = 7) after treatment with DHPG (82.6 ± 5.1, n = 9) or after 2𠄳 days of incubation with MCPG (66.5 ± 3.9, n = 11). Addition of puromycin simultaneously with either drug also did not significantly change the density of spines, although a trend toward fewer spines was seen with DHPG + puromycin (56.3 ± 1.0, n = 3 P = 0.06, Mann–Whitney U test, two-tailed).
To characterize further the nature of the changes induced by metabotropic receptor activation, the density of dendritic spines along secondary dendrites of dentate granule cells and dissociated hippocampal neurons was measured. Although the data suggested a small increase in the density with DHPG treatment (Fig. (Fig.5 5 B), these changes were not statistically significant and may have reflected the influence of increased length on our ability to discern existing postsynaptic elements.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Keywords: synapses, synaptic plasticity, hippocampus, super-resolution fluorescence microscopy, dendritic spines
Citation: Tønnesen J and Nägerl UV (2016) Dendritic Spines as Tunable Regulators of Synaptic Signals. Front. Psychiatry 7:101. doi: 10.3389/fpsyt.2016.00101
Received: 04 April 2016 Accepted: 27 May 2016
Published: 09 June 2016
Alberto A. Rasia-Filho, Federal University of Health Sciences, Brazil
Jon I. Arellano, Yale University School of Medicine, USA
Jochen Herms, German Center for Neurodegenerative Diseases (DZNE), Germany
Hjalmar Brismar, KTH Royal Institute of Technology, Sweden
Copyright: © 2016 Tønnesen and Nägerl. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.