Suspension of mitotic activity in dentate gyrus of the hibernating ground squirrel.

Neurogenesis occurs in the adult mammalian hippocampus, a region of the brain important for learning and memory. Hibernation in Siberian ground squirrels provides a natural model to study mitosis as the rapid fall in body temperature in 24 h (from 35-36°C to +4-6°C) permits accumulation of mitotic cells at different stages of the cell cycle. Histological methods used to study adult neurogenesis are limited largely to fixed tissue, and the mitotic state elucidated depends on the specific phase of mitosis at the time of day. However, using an immunohistochemical study of doublecortin (DCX) and BrdU-labelled neurons, we demonstrate that the dentate gyrus of the ground squirrel hippocampus contains a population of immature cells which appear to possess mitotic activity. Our data suggest that doublecortin-labelled immature cells exist in a mitotic state and may represent a renewable pool for generation of new neurons within the dentate gyrus.

Three-dimensional synaptic ultrastructure in the dentate gyrus and hippocampal area CA3 in the Ts65Dn mouse model of Down syndrome.

Down syndrome (DS) results from trisomy of human chromosome 21. Ts65Dn mice are an established model for DS and show several phenotypes similar to those in people with DS. However, there is little data on the structural plasticity of synapses in the trisynaptic pathway in the hippocampus. Here we investigate 3D ultrastructure of synapses in the hippocampus of age-matched control (2N) and Ts65Dn male mice. Serial ultrathin sections and 3D reconstructions characterize synapses in the middle molecular layer (MML) of dentate gyrus and in thorny excrescences (TEs) in proximal portions of apical dendrites of CA3 pyramidal neurons. 3D analysis of synapses shows phenotypes that distinguish Ts65Dn from 2N mice. For the MML, synapse density was reduced by 15% in Ts65Dn vs. 2N mice (P < 0.05). Comparative 3D analyses demonstrate a significant decrease in the number of thorns per TE in CA3 in Ts65Dn vs. 2N mice (by ≈45%, P = 0.01). Individual thorn volume was 3 times smaller in Ts65Dn vs. 2N mice (P = 0.02). A significant decrease in the number of thorn projections per TE in Ts65Dn vs. 2N mice was accompanied by a decrease of filopodium-like protrusions on the surface of TEs (P = 0.02). However, the volume of postsynaptic densities in CA3 Ts65Dn and 2N mice was unchanged (P = 0.78). Our findings suggest that the high degree of plasticity of CA3 thorns may be connected with their filopodial origin. Alterations of 3D synaptic structure in Ts65Dn mice may further contribute to the diminished plasticity in DS.

Complexity of contacts between synaptic boutons and dendritic spines in adult rat hippocampus: three-dimensional reconstructions from serial ultrathin sections in vivo.

The morphological relationship between a synapse and dendritic spine is an important determinant of synaptic function, and has been studied extensively with the electron microscope. This has revealed that the universal idea of one synapse per spine is no longer tenable; dendritic spines may on occasions form synapses with two or more axonal boutons. To elucidate the detailed structure and relationship of spines and their synaptic contacts in the molecular layers of the dentate gyrus of the adult rat, a study using 3-dimensional (3D) reconstructions was made from serial ultrathin sections (up to 150 sections per series), of both pre- and postsynaptic segments of axons and dendrites. We found that synapses from all unmyelinated axons examined were en passant and although most contacts between synapse and spine are one-to-one, all reconstructions examined contained both multisynaptic axonal segments, and multisynaptic spines. In the dentate gyrus, 3.48% mushroom spines were multisynaptic, synapsing with more than two axons, and 0.46% of the total population of synapses were multisynaptic. Although these values appear small, they actually comprise millions of contacts per cubic millimeter of tissue. Axonal varicosities can contact more than three dendritic spines originating from different dendritic branches while spines may branch with up to three heads, and spine heads may receive two or more synaptic contacts. Our data confirm the complexity of relationships between spines and synapses and show that a one-one relationship between synapse and spine cannot be assumed automatically.

Membrane lipids and morphology of brain cortex synaptosomes isolated from hibernating Yakutian ground squirrel.

Synaptosomes were isolated from Yakutian ground squirrel brain cortex of summer and winter hibernating animals in active and torpor states. Synaptosomal membrane cholesterol and phospholipids were determined. The seasonal changes of synaptosomal lipid composition were found. Synaptosomes isolated from hibernating Yakutian ground squirrel brain cortex maintained the cholesterol sphingomyelin, phosphatidylethanolamine, lysophosphatidylcholine, cardiolipin, phosphatidylinositol and phosphatidylserine contents 2.5, 1.8, 2.6, 1.8, 1.6, and 1.3 times less, respectively, and the content of phosphatidylcholine twice as much as the one in summer season. The synaptosomal membrane lipid composition of summer animals was shown to be markedly different from that as hibernating ground squirrels and non-hibernating rodents. It is believed that phenotypic changes of synaptosomal membrane lipid composition in summer Yakutian ground squirrel are the important preparation step for hibernation. The phosphatidylethanolamine content was increased in torpor state compared with winter-active state and the molar ratio of cholesterol/phospholipids in synaptosomal membrane of winter torpor ground squirrels was lower than that in active winter and summer animals. These events were supposed to lead to increase of the synaptosomal membrane fluidity during torpor. Synaptosomes isolated from torpor animals have larger sizes and contain a greater number of synaptic vesicles on the synaptosomal profile area. The synaptosomal membrane lipid composition and synaptosome morphology were involved in phenotypic adaptation of Yakutian ground squirrel to hibernation.

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 microm. 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 approximately 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.

The glutamate receptor 2 subunit controls post-synaptic density complexity and spine shape in the dentate gyrus.

In adult brain the majority of AMPA glutamate receptor (GluR) subunits contain GluR2. In knock-out (KO) mice the absence of GluR2 results in consequences for synaptic plasticity including cognitive impairments. Here the morphology of dendritic spines and their synaptic contacts was analysed via three-dimensional reconstruction of serial electron micrographs from dentate gyrus (DG) of adult wild type (WT) and GluR2 KO mice. Pre-embedding immunocytochemical staining was used to examine the distribution and subcellular localization of AMPA receptor GluR1 and N-methyl-D-aspartate receptor NR1 subunits. There were no significant changes in synapse density in the DG of GluR2 KO compared with WT mice. However, in GluR2 KO mice there was a significant decrease in the percentage of synapses on mushroom spines but an increase in synapses on thin spines. There was also a large decrease in the proportion of synapses with complex perforated/segmented post-synaptic densities (PSDs) (25 vs. 78% in WT) but an increase in synapses with macular PSDs (75 vs. 22%). These data were coupled in GluR2 KO mice with significant decreases in volume and surface area of mushroom spines and their PSDs. In both GluR2 KO and WT mice, NR1 and GluR1 receptors were present in dendrites and spines but there was a significant reduction in NR1 labelling of spine membranes and cytoplasm in GluR2 KO mice, and a small decrease in GluR1 immunolabelling in membranes and cytoplasm of spines in GluR2 KO compared with WT mice. Our data demonstrate that the absence of GluR2 has a significant effect on both DG synapse and spine cytoarchitecture and the expression of NR1 receptors.

Expression of long-term potentiation in aged rats involves perforated synapses but dendritic spine branching results from high-frequency stimulation alone.

Evidence for morphological substrates of long-term changes in synaptic efficacy is controversial, partly because it is difficult to employ an unambiguous control. We have used a high-frequency stimulation protocol in vivo to induce long-term potentiation (LTP) in the hippocampal dentate gyrus of aged (22-month-old) rats and have found a clear distinction between animals that sustain LTP and those that fail to sustain it. The "failure group" was used as a specific/"like-with-like" control for morphological changes associated with the expression of LTP per se. Quantitative optical and electron microscopy was used to analyze large populations of dendritic spines and excitatory perforant path synapses; LTP was found to be associated with an increase in numbers of segmented (perforated) postsynaptic densities in spine synapses. In contrast, an increase in the number of branched spines appears to result from high-frequency stimulation alone. These data shed light on the current controversy about the expression mechanism of LTP.

Rapid reversal of stress induced loss of synapses in CA3 of rat hippocampus following water maze training.

The impact was examined of exposing rats to two life experiences of a very different nature (stress and learning) on synaptic structures in hippocampal area CA3. Rats were subjected to either (i) chronic restraint stress for 21 days, and/or (ii) spatial training in a Morris water maze. At the behavioural level, restraint stress induced an impairment of acquisition of the spatial response. Moreover, restraint stress and water maze training had contrasting impacts on CA3 synaptic morphometry. Chronic stress induced a loss of simple asymmetric synapses [those with an unperforated postsynaptic density (PSD)], whilst water maze learning reversed this effect, promoting a rapid recovery of stress-induced synaptic loss within 2-3 days following stress. In addition, in unstressed animals a correlation was found between learning efficiency and the density of synapses with an unperforated PSD: the better the performance in the water maze, the lower the synaptic density. Water maze training increased the number of perforated synapses (those with a segmented PSD) in CA3, both in stressed and, more notably, in unstressed rats. The distinct effects of stress and learning on CA3 synapses reported here provide a neuroanatomical basis for the reported divergent effects of these experiences on hippocampal synaptic activity, i.e. stress as a suppressor and learning as a promoter of synaptic plasticity.