Impaired synaptic incorporation of AMPA receptors in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is the most common monogenetic cause of inherited intellectual disability and autism in humans. One of the well-characterized molecular phenotypes of Fmr1 KO mice, a model of FXS, is increased translation of synaptic proteins. Although this upregulation stabilizes in adulthood, abnormalities during the critical period of plasticity have long-term effects on circuit formation and synaptic properties. Using high-resolution quantitative proteomics of synaptoneurosomes isolated from the adult, developed brains of Fmr1 KO mice, we show a differential abundance of proteins regulating the postsynaptic receptor activity of glutamatergic synapses. We investigated the AMPA receptor composition and shuttling in adult Fmr1 KO and WT mice using a variety of complementary experimental strategies such as surface protein crosslinking, immunostaining of surface receptors, and electrophysiology. We discovered that the activity-dependent synaptic delivery of AMPARs is impaired in adult Fmr1 KO mice. Furthermore, we show that Fmr1 KO synaptic AMPARs contain more GluA2 subunits that can be interpreted as a switch in the synaptic AMPAR subtype toward an increased number of Ca2+-impermeable receptors in adult Fmr1 KO synapses.

Disrupting interaction between miR-132 and Mmp9 3'UTR improves synaptic plasticity and memory in mice.

As microRNAs have emerged to be important regulators of molecular events occurring at the synapses, the new questions about their regulatory effect on the behavior have araised. In the present study, we show for the first time that the dysregulated specific targeting of miR132 to Mmp9 mRNA in the mouse brain results in the increased level of Mmp9 protein, which affects synaptic plasticity and has an effect on memory formation. Our data points at the importance of complex and precise regulation of the Mmp9 level by miR132 in the brain.

Activation of the 5-HT7 receptor and MMP-9 signaling module in the hippocampal CA1 region is necessary for the development of depressive-like behavior.

Major depressive disorder is a complex disease resulting from aberrant synaptic plasticity that may be caused by abnormal serotonergic signaling. Using a combination of behavioral, biochemical, and imaging methods, we analyze 5-HT7R/MMP-9 signaling and dendritic spine plasticity in the hippocampus in mice treated with the selective 5-HT7R agonist (LP-211) and in a model of chronic unpredictable stress (CUS)-induced depressive-like behavior. We show that acute 5-HT7R activation induces depressive-like behavior in mice in an MMP-9-dependent manner and that post mortem brain samples from human individuals with depression reveal increased MMP-9 enzymatic activity in the hippocampus. Both pharmacological activation of 5-HT7R and modulation of its downstream effectors as a result of CUS lead to dendritic spine elongation and decreased spine density in this region. Overall, the 5-HT7R/MMP-9 pathway is specifically activated in the CA1 subregion of the hippocampus during chronic stress and is crucial for inducing depressive-like behavior.

The cell adhesion protein dystroglycan affects the structural remodeling of dendritic spines.

Dystroglycan (DG) is a cell membrane protein that binds to the extracellular matrix in various mammalian tissues. The function of DG has been well defined in embryonic development as well as in the proper migration of differentiated neuroblasts in the central nervous system (CNS). Although DG is known to be a target for matrix metalloproteinase-9 (MMP-9), cleaved in response to enhanced synaptic activity, the role of DG in the structural remodeling of dendritic spines is still unknown. Here, we report for the first time that the deletion of DG in rat hippocampal cell cultures causes pronounced changes in the density and morphology of dendritic spines. Furthermore, we noted a decrease in laminin, one of the major extracellular partners of DG. We have also observed that the lack of DG evokes alterations in the morphological complexity of astrocytes accompanied by a decrease in the level of aquaporin 4 (AQP4), a protein located within astrocyte endfeet surrounding neuronal dendrites and synapses. Regardless of all of these changes, we did not observe any effect of DG silencing on either excitatory or inhibitory synaptic transmission. Likewise, the knockdown of DG had no effect on Psd-95 protein expression. Our results indicate that DG is involved in dendritic spine remodeling that is not functionally reflected. This may suggest the existence of unknown mechanisms that maintain proper synaptic signaling despite impaired structure of dendritic spines. Presumably, astrocytes are involved in these processes.

Three-Dimensional Segmentation and Reconstruction of Neuronal Nuclei in Confocal Microscopic Images.

The detailed architectural examination of the neuronal nuclei in any brain region, using confocal microscopy, requires quantification of fluorescent signals in three-dimensional stacks of confocal images. An essential prerequisite to any quantification is the segmentation of the nuclei which are typically tightly packed in the tissue, the extreme being the hippocampal dentate gyrus (DG), in which nuclei frequently appear to overlap due to limitations in microscope resolution. Segmentation in DG is a challenging task due to the presence of a significant amount of image artifacts and densely packed nuclei. Accordingly, we established an algorithm based on continuous boundary tracing criterion aiming to reconstruct the nucleus surface and to separate the adjacent nuclei. The presented algorithm neither uses a pre-built nucleus model, nor performs image thresholding, which makes it robust against variations in image intensity and poor contrast. Further, the reconstructed surface is used to study morphology and spatial arrangement of the nuclear interior. The presented method is generally dedicated to segmentation of crowded, overlapping objects in 3D space. In particular, it allows us to study quantitatively the architecture of the neuronal nucleus using confocal-microscopic approach.

Prophylactic Ketamine Treatment Promotes Resilience to Chronic Stress and Accelerates Recovery: Correlation with Changes in Synaptic Plasticity in the CA3 Subregion of the Hippocampus.

Ketamine is an N-methyl-d-aspartate receptor antagonist that has gained wide attention as a potent antidepressant. It has also been recently reported to have prophylactic effects in animal models of depression and anxiety. Alterations of neuroplasticity in different brain regions; such as the hippocampus; prefrontal cortex; and amygdala; are a hallmark of stress-related disorders; and such changes may endure beyond the treatment of symptoms. The present study investigated whether a prophylactic injection of ketamine has effects on structural plasticity in the brain in mice that are subjected to chronic unpredictable stress followed by an 8-day recovery period. Ketamine administration (3 mg/kg body weight) 1 h before stress exposure increased the number of resilient animals immediately after the cessation of stress exposure and positively influenced the recovery of susceptible animals to hedonic deficits. At the end of the recovery period; ketamine-treated animals exhibited significant differences in dendritic spine density and dendritic spine morphology in brain regions associated with depression compared with saline-treated animals. These results confirm previous findings of the prophylactic effects of ketamine and provide further evidence of an association between the antidepressant-like effect of ketamine and alterations of structural plasticity in the brain.

Author Correction: Quantitative 3-D morphometric analysis of individual dendritic spines.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Quantitative 3-D morphometric analysis of individual dendritic spines.

The observation and analysis of dendritic spines morphological changes poses a major challenge in neuroscience studies. The alterations of their density and/or morphology are indicators of the cellular processes involved in neural plasticity underlying learning and memory, and are symptomatic in neuropsychiatric disorders. Despite ongoing intense investigations in imaging approaches, the relationship between changes in spine morphology and synaptic function is still unknown. The existing quantitative analyses are difficult to perform and require extensive user intervention. Here, we propose a new method for (1) the three-dimensional (3-D) segmentation of dendritic spines using a multi-scale opening approach and (2) define 3-D morphological attributes of individual spines for the effective assessment of their structural plasticity. The method was validated using confocal light microscopy images of dendritic spines from dissociated hippocampal cultures and brain slices (1) to evaluate accuracy relative to manually labeled ground-truth annotations and relative to the state-of-the-art Imaris tool, (2) to analyze reproducibility of user-independence of the segmentation method, and (3) to quantitatively analyze morphological changes in individual spines before and after chemically induced long-term potentiation. The method was monitored and used to precisely describe the morphology of individual spines in real-time using consecutive images of the same dendritic fragment.

Transient ECM protease activity promotes synaptic plasticity.

Activity-dependent proteolysis at a synapse has been recognized as a pivotal factor in controlling dynamic changes in dendritic spine shape and function; however, excessive proteolytic activity is detrimental to the cells. The exact mechanism of control of these seemingly contradictory outcomes of protease activity remains unknown. Here, we reveal that dendritic spine maturation is strictly controlled by the proteolytic activity, and its inhibition by the endogenous inhibitor (Tissue inhibitor of matrix metalloproteinases-1 - TIMP-1). Excessive proteolytic activity impairs long-term potentiation of the synaptic efficacy (LTP), and this impairment could be rescued by inhibition of protease activity. Moreover LTP is altered persistently when the ability of TIMP-1 to inhibit protease activity is abrogated, further demonstrating the role of such inhibition in the promotion of synaptic plasticity under well-defined conditions. We also show that dendritic spine maturation involves an intermediate formation of elongated spines, followed by their conversion into mushroom shape. The formation of mushroom-shaped spines is accompanied by increase in AMPA/NMDA ratio of glutamate receptors. Altogether, our results identify inhibition of protease activity as a critical regulatory mechanism for dendritic spines maturation.

2dSpAn: semiautomated 2-d segmentation, classification and analysis of hippocampal dendritic spine plasticity.

MOTIVATION: Accurate and effective dendritic spine segmentation from the dendrites remains as a challenge for current neuroimaging research community. In this article, we present a new method (2dSpAn) for 2-d segmentation, classification and analysis of structural/plastic changes of hippocampal dendritic spines. A user interactive segmentation method with convolution kernels is designed to segment the spines from the dendrites. Formal morphological definitions are presented to describe key attributes related to the shape of segmented spines. Spines are automatically classified into one of four classes: Stubby, Filopodia, Mushroom and Spine-head Protrusions. RESULTS: The developed method is validated using confocal light microscopy images of dendritic spines from dissociated hippocampal cultures for: (i) quantitative analysis of spine morphological changes, (ii) reproducibility analysis for assessment of user-independence of the developed software and (iii) accuracy analysis with respect to the manually labeled ground truth images, and also with respect to the available state of the art. The developed method is monitored and used to precisely describe the morphology of individual spines in real-time experiments, i.e. consequent images of the same dendritic fragment. AVAILABILITY AND IMPLEMENTATION: The software and the source code are available at https://sites.google.com/site/2dspan/ under open-source license for non-commercial use. CONTACT: subhadip@cse.jdvu.ac.in or j.wlodarczyk@nencki.gov.pl SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Computational Approach to Dendritic Spine Taxonomy and Shape Transition Analysis.

The common approach in morphological analysis of dendritic spines of mammalian neuronal cells is to categorize spines into subpopulations based on whether they are stubby, mushroom, thin, or filopodia shaped. The corresponding cellular models of synaptic plasticity, long-term potentiation, and long-term depression associate the synaptic strength with either spine enlargement or spine shrinkage. Although a variety of automatic spine segmentation and feature extraction methods were developed recently, no approaches allowing for an automatic and unbiased distinction between dendritic spine subpopulations and detailed computational models of spine behavior exist. We propose an automatic and statistically based method for the unsupervised construction of spine shape taxonomy based on arbitrary features. The taxonomy is then utilized in the newly introduced computational model of behavior, which relies on transitions between shapes. Models of different populations are compared using supplied bootstrap-based statistical tests. We compared two populations of spines at two time points. The first population was stimulated with long-term potentiation, and the other in the resting state was used as a control. The comparison of shape transition characteristics allowed us to identify the differences between population behaviors. Although some extreme changes were observed in the stimulated population, statistically significant differences were found only when whole models were compared. The source code of our software is freely available for non-commercial use. CONTACT: d.plewczynski@cent.uw.edu.pl.