Combined hyperforin and lanicemine treatment instead of ketamine or imipramine restores behavioral deficits induced by chronic restraint stress and dietary zinc restriction in mice.

Clinical and preclinical studies show evidence that chronic stress or nutritional deficits in dietary zinc (Zn) intake may be risk factors for developing major depressive disorder (MDD). Furthermore, there may be possible links between low serum Zn levels and development of treatment-resistant depression. In the present work, we combined chronic restraint stress (CRS) and a low-zinc diet (ZnD) in mice and carried out a set of behavioral and biochemical studies. The mice were treated with four different antidepressant compounds, namely, ketamine, Ro 25-6981 (Ro), hyperforin and lanicemine (Hyp + Lan), and imipramine (IMI). We show that CRS or ZnD alone or a combination of CRS and ZnD (CRS + ZnD) induces anhedonia observed in the sucrose preference test (SPT). The behavioral effects of CRS were restored by ketamine or IMI. However, only Hyp + Lan restored the deficits in behavioral phenotype in mice subjected to CRS + ZnD. We also showed that the antidepressant-like effects observed in Hyp + Lan-treated CRS + ZnD mice were associated with changes in the morphology of the dendritic spines (restored physiological level) in the hippocampus (Hp). Finally, we studied the metabolism of ketamine and its brain absorption in CRS and CRS + ZnD mice. Our results suggest that CRS + ZnD does not alter the metabolism of ketamine to (2R,6R;2S,6S)-HNK; however, CRS + ZnD can induce altered bioavailability and distribution of ketamine in the Hp and frontal cortex (FC) in CRS + ZnD animals compared to the control and CRS groups.

SRF depletion in early life contributes to social interaction deficits in the adulthood.

Alterations in social behavior are core symptoms of major developmental neuropsychiatric diseases such as autism spectrum disorders or schizophrenia. Hence, understanding their molecular and cellular underpinnings constitutes the major research task. Dysregulation of the global gene expression program in the developing brain leads to modifications in a number of neuronal connections, synaptic strength and shape, causing unbalanced neuronal plasticity, which may be important substrate in the pathogenesis of neurodevelopmental disorders, contributing to their clinical outcome. Serum response factor (SRF) is a major transcription factor in the brain. The behavioral influence of SRF deletion during neuronal differentiation and maturation has never been studied because previous attempts to knock-out the gene caused premature death. Herein, we generated mice that lacked SRF from early postnatal development to precisely investigate the role of SRF starting in the specific time window before maturation of excitatory synapses that are located on dendritic spine occurs. We show that the time-controlled loss of SRF in neurons alters specific aspects of social behaviors in SRF knock-out mice, and causes deficits in developmental spine maturation at both the structural and functional levels, including downregulated expression of the AMPARs subunits GluA1 and GluA2, and increases the percentage of filopodial/immature dendritic spines. In aggregate, our study uncovers the consequences of postnatal SRF elimination for spine maturation and social interactions revealing novel mechanisms underlying developmental neuropsychiatric diseases.

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.

S-Palmitoylation of Synaptic Proteins as a Novel Mechanism Underlying Sex-Dependent Differences in Neuronal Plasticity.

Although sex differences in the brain are prevalent, the knowledge about mechanisms underlying sex-related effects on normal and pathological brain functioning is rather poor. It is known that female and male brains differ in size and connectivity. Moreover, those differences are related to neuronal morphology, synaptic plasticity, and molecular signaling pathways. Among different processes assuring proper synapse functions are posttranslational modifications, and among them, S-palmitoylation (S-PALM) emerges as a crucial mechanism regulating synaptic integrity. Protein S-PALM is governed by a family of palmitoyl acyltransferases, also known as DHHC proteins. Here we focused on the sex-related functional importance of DHHC7 acyltransferase because of its S-PALM action over different synaptic proteins as well as sex steroid receptors. Using the mass spectrometry-based PANIMoni method, we identified sex-dependent differences in the S-PALM of synaptic proteins potentially involved in the regulation of membrane excitability and synaptic transmission as well as in the signaling of proteins involved in the structural plasticity of dendritic spines. To determine a mechanistic source for obtained sex-dependent changes in protein S-PALM, we analyzed synaptoneurosomes isolated from DHHC7-/- (DHHC7KO) female and male mice. Our data showed sex-dependent action of DHHC7 acyltransferase. Furthermore, we revealed that different S-PALM proteins control the same biological processes in male and female synapses.

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.

A dominant dendrite phenotype caused by the disease-associated G253D mutation in doublecortin (DCX) is not due to its endocytosis defect.

Doublecortin (DCX) is a protein needed for cortical development, and DCX mutations cause cortical malformations in humans. The microtubule-binding activity of DCX is well-described and is important for its function, such as supporting neuronal migration and dendrite growth during development. Previous work showed that microtubule binding is not sufficient for DCX-mediated promotion of dendrite growth and that domains in DCX's C terminus are also required. The more C-terminal regions of DCX bind several other proteins, including the adhesion receptor neurofascin and clathrin adaptors. We recently identified a role for DCX in endocytosis of neurofascin. The disease-associated DCX-G253D mutant protein is known to be deficient in binding neurofascin, and we now asked if disruption of neurofascin endocytosis underlies the DCX-G253D-associated pathology. We first demonstrated that DCX functions in endocytosis as a complex with both the clathrin adaptor AP-2 and neurofascin: disrupting either clathrin adaptor binding (DCX-ALPA) or neurofascin binding (DCX-G253D) decreased neurofascin endocytosis in primary neurons. We then investigated a known function for DCX, namely, increasing dendrite growth in cultured neurons. Surprisingly, we found that the DCX-ALPA and DCX-G253D mutants yield distinct dendrite phenotypes. Unlike DCX-ALPA, DCX-G253D caused a dominant-negative dendrite growth phenotype. The endocytosis defect of DCX-G253D thus was separable from its detrimental effects on dendrite growth. We recently identified Dcx-R59H as a dominant allele and can now classify Dcx-G253D as a second Dcx allele that acts dominantly to cause pathology, but does so via a different mechanism.

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.

CD44: a novel synaptic cell adhesion molecule regulating structural and functional plasticity of dendritic spines.

Synaptic cell adhesion molecules regulate signal transduction, synaptic function, and plasticity. However, their role in neuronal interactions with the extracellular matrix (ECM) is not well understood. Here we report that the CD44, a transmembrane receptor for hyaluronan, modulates synaptic plasticity. High-resolution ultrastructural analysis showed that CD44 was localized at mature synapses in the adult brain. The reduced expression of CD44 affected the synaptic excitatory transmission of primary hippocampal neurons, simultaneously modifying dendritic spine shape. The frequency of miniature excitatory postsynaptic currents decreased, accompanied by dendritic spine elongation and thinning. These structural and functional alterations went along with a decrease in the number of presynaptic Bassoon puncta, together with a reduction of PSD-95 levels at dendritic spines, suggesting a reduced number of functional synapses. Lack of CD44 also abrogated spine head enlargement upon neuronal stimulation. Moreover, our results indicate that CD44 contributes to proper dendritic spine shape and function by modulating the activity of actin cytoskeleton regulators, that is, Rho GTPases (RhoA, Rac1, and Cdc42). Thus CD44 appears to be a novel molecular player regulating functional and structural plasticity of dendritic spines.

Different Doublecortin (DCX) Patient Alleles Show Distinct Phenotypes in Cultured Neurons: EVIDENCE FOR DIVERGENT LOSS-OF-FUNCTION AND "OFF-PATHWAY" CELLULAR MECHANISMS.

Doublecortin on the X-chromosome (DCX) is a neuronal microtubule-binding protein with a multitude of roles in neurodevelopment. In humans, DCX is a major genetic locus for X-linked lissencephaly. The best studied defects are in neuronal migration during corticogenesis and in the hippocampus, as well as axon and dendrite growth defects. Much effort has been directed at understanding the molecular and cellular bases of DCX-linked lissencephaly. The focus has been in particular on defects in microtubule assembly and bundling, using knock-out mice and expression of WT and mutant Dcx in non-neuronal cells. Dcx also binds other proteins besides microtubules, such as spinophilin (abbreviated spn; gene name Ppp1r9b protein phosphatase 1 regulatory subunit 9b) and the clathrin adaptors AP-1 and AP-2. Even though many non-sense and missense mutations of Dcx are known, their molecular and cellular defects are still only incompletely understood. It is also largely unknown how neurons are affected by expression of DCX patient alleles. We have now characterized several patient DCX alleles (DCX-R89G, DCX-R59H, DCX-246X, DCX-272X, and DCX-303X) using a gain-of-function dendrite growth assay in cultured rat neurons in combination with the determination of molecular binding activities and subcellular localization in non-neuronal and neuronal cells. First, we find that several mutants (Dcx-R89G and Dcx-272X) were loss-of-function alleles (as had been postulated) but surprisingly acted via different cellular mechanisms. Second, one allele (Dcx-R59H) formed cytoplasmic aggregates, which contained Hspa1B (heat shock protein 1B hsp70) and ubiquitinated proteins, trapped other cytoskeletal proteins, including spinophilin, and led to increased autophagy. This allele could thus be categorized as "off-pathway"/possibly neomorph. Our findings thus suggested that distinct DCX alleles caused dysfunction by different mechanisms.

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.