Igor Kraev

Stress suppresses and learning induces plasticity in CA3 of rat hippocampus: a three-dimensional ultrastructural study of thorny excrescences and their postsynaptic densities

Chronic stress and spatial training have been proposed to affect hippocampal structure and function in opposite ways. Previous morphological studies that addressed structural changes after chronic restraint stress and spatial training were based on two-dimensional morphometry which does not allow a complete morphometric characterisation of synaptic features. Here, for the first time in such studies, we examined these issues by using three-dimensional (3-D) reconstructions of electron microscope images taken from thorny excrescences of hippocampal CA3 pyramidal cells. Ultrastructural alterations in postsynaptic densities (PSDs) of thorny excrescences receiving input from mossy fibre boutons were also determined, as were changes in numbers of multivesicular bodies (endosome-like structures) within thorny excrescences and dendrites. Quantitative 3-D data demonstrated retraction of thorny excrescences after chronic restraint stress which was reversed after water maze training, whilst water maze training alone increased thorny excrescence volume and number of thorns per thorny excrescence. PSD surface area was unaffected by restraint stress but water maze training increased both number and area of PSDs per thorny excrescence. In restrained rats that were water maze trained PSD volume and surface area increased significantly. The proportion of perforated PSDs almost doubled after water maze training and restraint stress. Numbers of endosome-like structures in thorny excrescences decreased after restraint stress and increased after water maze training. These findings demonstrate that circuits involving contacts between mossy fibre terminals and CA3 pyramidal cells at stratum lucidum level are affected conversely by water maze training and chronic stress, confirming the remarkable plasticity of CA3 dendrites. They provide a clear illustration of the structural modifications that occur after life experiences noted for their different impact on hippocampal function.

Forebrain CRF 1 modulates early-life stress-programmed cognitive deficits

Childhood traumatic events hamper the development of the hippocampus and impair declarative memory in susceptible individuals. Persistent elevations of hippocampal corticotropin-releasing factor (CRF), acting through CRF receptor 1 (CRF1), in experimental models of early-life stress have suggested a role for this endogenous stress hormone in the resulting structural modifications and cognitive dysfunction. However, direct testing of this possibility has been difficult. In the current study, we subjected conditional forebrain CRF1 knock-out (CRF1-CKO) mice to an impoverished postnatal environment and examined the role of forebrain CRF1 in the long-lasting effects of early-life stress on learning and memory. Early-life stress impaired spatial learning and memory in wild-type mice, and postnatal forebrain CRF overexpression reproduced these deleterious effects. Cognitive deficits in stressed wild-type mice were associated with disrupted long-term potentiation (LTP) and a reduced number of dendritic spines in area CA3 but not in CA1. Forebrain CRF1 deficiency restored cognitive function, LTP and spine density in area CA3, and augmented CA1 LTP and spine density in stressed mice. In addition, early-life stress differentially regulated the amount of hippocampal excitatory and inhibitory synapses in wild-type and CRF1-CKO mice, accompanied by alterations in the neurexin-neuroligin complex. These data suggest that the functional, structural and molecular changes evoked by early-life stress are at least partly dependent on persistent forebrain CRF1 signaling, providing a molecular target for the prevention of cognitive deficits in adults with a history of early-life adversity.

Chemically induced long‐term potentiation increases the number of perforated and complex postsynaptic densities but does not alter dendritic spine volume in CA1 of adult mouse hippocampal slices

Examination of the morphological correlates of long‐term potentiation (LTP) in the hippocampus requires the analysis of both the presynaptic and postsynaptic elements. However, ultrastructural measurements of synapses and dendritic spines following LTP induced via tetanic stimulation presents the difficulty that not all synapses examined are necessarily activated. To overcome this limitation, and to ensure that a very large proportion of the synapses and spines examined have been potentiated, we induced LTP in acute hippocampal slices of adult mice by addition of tetraethylammonium (TEA) to a modified CSF containing an elevated concentration of Ca2+ and no Mg+. Quantitative electron microscope morphometric analyses and three‐dimensional (3‐D) reconstructions of both dendritic spines and postsynaptic densities (PSDs) in CA1 stratum radiatum were made on serial ultrathin sections. One hour after chemical LTP induction the proportion of macular (unperforated) synapses decreased (50%) whilst the number of synapses with simple perforated and complex PSDs (nonmacular) increased significantly (17%), without significant changes in volume and surface area of the PSD. In addition, the surface area of mushroom spines increased significantly (13%) whilst there were no volume differences in either mushroom or thin spines, or in surface area of thin spines. CA1 stratum radiatum contained multiple‐synapse en passant axons as well as multiple‐synapse spines, which were unaffected by chemical LTP. Our results suggest that chemical LTP induces active dendritic spine remodelling and correlates with a change in the weight and strength of synaptic transmission as shown by the increase in the proportion of nonmacular synapses.

A cell adhesion molecule mimetic, FGL peptide, induces alterations in synapse and dendritic spine structure in the dentate gyrus of aged rats: a three-dimensional ultrastructural study

The FGL peptide is a neural cell adhesion molecule (NCAM) mimetic comprising a 15‐amino‐acid‐long sequence of the FG loop region of the second fibronectin type III module of NCAM. It corresponds to the binding site of NCAM for the fibroblast growth factor receptor 1. FGL improves cognitive function through enhancement of synaptic function. We examined the effect of FGL on synaptic and dendritic structure in the brains of aged (22‐month‐old) rats that were injected subcutaneously (8 mg/kg) at 2‐day intervals until 19 days after the start of the experiment. Animals were perfused with fixative, brains removed and coronal sections cut at 50 µm. The hippocampal volume was measured, tissue embedded and ultrathin sections viewed in a JEOL 1010 electron microscope. Analyses were made of synaptic and dendritic parameters following three‐dimensional reconstruction via images from a series of ∼100 serial ultrathin sections. FGL affected neither hippocampal volume nor spine or synaptic density in the middle molecular layer of the dentate gyrus. However, it increased the ratio of mushroom to thin spines, number of multivesicular bodies and also increased the frequency of appearance of coated pits. Three‐dimensional analysis showed a significant decrease in both post‐synaptic density and apposition zone curvature of mushroom spines following FGL treatment, whereas for thin spines the convexity of the apposition zone increased. These data indicate that FGL induces large changes in the fine structure of synapses and dendritic spines in hippocampus of aged rats, complementing data showing its effect on cognitive processes.

Impaired hippocampal neuroligin-2 function by chronic stress or synthetic peptide treatment is linked to social deficits and increased aggression

Neuroligins (NLGNs) are cell adhesion molecules that are important for proper synaptic formation and functioning, and are critical regulators of the balance between neural excitation/inhibition (E/I). Mutations in NLGNs have been linked to psychiatric disorders in humans involving social dysfunction and are related to similar abnormalities in animal models. Chronic stress increases the likelihood for affective disorders and has been shown to induce changes in neural structure and function in different brain regions, with the hippocampus being highly vulnerable to stress. Previous studies have shown evidence of chronic stress-induced changes in the neural E/I balance in the hippocampus. Therefore, we hypothesized that chronic restraint stress would lead to reduced hippocampal NLGN-2 levels, in association with alterations in social behavior. We found that rats submitted to chronic restraint stress in adulthood display reduced sociability and increased aggression. This occurs along with a reduction of NLGN-2, but not NLGN-1 expression (as shown with western blot, immunohistochemistry, and electron microscopy analyses), throughout the hippocampus and detectable in different layers of the CA1, CA3, and DG subfields. Furthermore, using synthetic peptides that comprise sequences in either NLGN-1 (neurolide-1) or NLGN-2 (neurolide-2) involved in the interaction with their presynaptic partner neurexin (NRXN)-1, intra-hippocampal administration of neurolide-2 led also to reduced sociability and increased aggression. These results highlight hippocampal NLGN-2 as a key molecular substrate regulating social behaviors and underscore NLGNs as promising targets for the development of novel drugs for the treatment of dysfunctional social behaviors.

Glia selectively approach synapses on thin dendritic spines

This paper examines the relationship between the morphological modality of 189 dendritic spines and the surrounding astroglia using full three-dimensional reconstructions of neuropil fragments. An integrative measure of three-dimensional glial coverage confirms that thin spine postsynaptic densities are more tightly surrounded by glia. This distinction suggests that diffusion-dependent synapse–glia communication near ‘learning’ synapses (associated with thin spines) could be stronger than that near ‘memory’ synapses (associated with larger spines).

The N-methyl-d-aspartate receptor antagonist CPP alters synapse and spine structure and impairs long-term potentiation and long-term depression induced morphological plasticity in dentate gyrus of the awake rat

Long-term morphological synaptic changes associated with homosynaptic long-term potentiation (LTP) and heterosynaptic long-term depression (LTD) in vivo, in awake adult rats were analyzed using three-dimensional (3-D) reconstructions of electron microscope images of ultrathin serial sections from the molecular layer of the dentate gyrus. For the first time in morphological studies, the specificity of the effects of LTP and LTD on both spine and synapse ultrastructure was determined using an N-methyl-d-aspartate (NMDA) receptor antagonist CPP (3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid). There were no differences in synaptic density 24 h after LTP or LTD induction, and CPP alone had no effect on synaptic density. LTP increased significantly the proportion of mushroom spines, whereas LTD increased the proportion of thin spines, and both LTP and LTD decreased stubby spine number. Both LTP and LTD increased significantly spine head evaginations (spinules) into synaptic boutons and CPP blocked these changes. Synaptic boutons were smaller after LTD, indicating a pre-synaptic effect. Interestingly, CPP alone decreased bouton and mushroom spine volumes, as well as post-synaptic density (PSD) volume of mushroom spines.These data show similarities, but also some clear differences, between the effects of LTP and LTD on spine and synaptic morphology. Although CPP blocks both LTP and LTD, and impairs most morphological changes in spines and synapses, CPP alone was shown to exert effects on aspects of spine and synaptic structure.

A Peptide Mimetic Targeting Trans-Homophilic NCAM Binding Sites Promotes Spatial Learning and Neural Plasticity in the Hippocampus

The key roles played by the neural cell adhesion molecule (NCAM) in plasticity and cognition underscore this membrane protein as a relevant target to develop cognitive-enhancing drugs. However, NCAM is a structurally and functionally complex molecule with multiple domains engaged in a variety of actions, which raise the question as to which NCAM fragment should be targeted. Synthetic NCAM mimetic peptides that mimic NCAM sequences relevant to specific interactions allow identification of the most promising targets within NCAM. Recently, a decapeptide ligand of NCAM—plannexin, which mimics a homophilic trans-binding site in Ig2 and binds to Ig3—was developed as a tool for studying NCAMs trans-interactions. In this study, we investigated plannexins ability to affect neural plasticity and memory formation. We found that plannexin facilitates neurite outgrowth in primary hippocampal neuronal cultures and improves spatial learning in rats, both under basal conditions and under conditions involving a deficit in a key plasticity-promoting posttranslational modification of NCAM, its polysialylation. We also found that plannexin enhances excitatory synaptic transmission in hippocampal area CA1, where it also increases the number of mushroom spines and the synaptic expression of the AMPAR subunits GluA1 and GluA2. Altogether, these findings provide compelling evidence that plannexin is an important facilitator of synaptic functional, structural and molecular plasticity in the hippocampal CA1 region, highlighting the fragment in NCAMs Ig3 module where plannexin binds as a novel target for the development of cognition-enhancing drugs.

Hippocampal circuit dysfunction in the Tc1 mouse model of Down syndrome

Hippocampal pathology is likely to contribute to cognitive disability in Down syndrome, yet the neural network basis of this pathology and its contributions to different facets of cognitive impairment remain unclear. Here we report dysfunctional connectivity between dentate gyrus and CA3 networks in the transchromosomic Tc1 mouse model of Down syndrome, demonstrating that ultrastructural abnormalities and impaired short-term plasticity at dentate gyrus–CA3 excitatory synapses culminate in impaired coding of new spatial information in CA3 and CA1 and disrupted behavior in vivo. These results highlight the vulnerability of dentate gyrus–CA3 networks to aberrant human chromosome 21 gene expression and delineate hippocampal circuit abnormalities likely to contribute to distinct cognitive phenotypes in Down syndrome.

Early Structural and Functional Defects in Synapses and Myelinated Axons in Stratum Lacunosum Moleculare in Two Preclinical Models for Tauopathy

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer’s disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3β with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer’s disease.