Multiple spine boutons are formed after long-lasting LTP in the awake rat.

The formation of multiple spine boutons (MSBs) has been associated with cognitive abilities including hippocampal-dependent associative learning and memory. Data obtained from cultured hippocampal slices suggest that the long-term maintenance of synaptic plasticity requires the formation of new synaptic contacts on pre-existing synapses. This postulate however, has never been tested in the awake, freely moving animals. In the current study, we induced long-term potentiation (LTP) in the dentate gyrus (DG) of awake adult rats and performed 3-D reconstructions of electron micrographs from thin sections of both axonal boutons and dendritic spines, 24 h post-induction. The specificity of the observed changes was demonstrated by comparison with animals in which long-term depression (LTD) had been induced, or with animals in which LTP was blocked by an N-methyl-D-aspartate (NMDA) antagonist. Our data demonstrate that whilst the number of boutons remains unchanged, there is a marked increase in the number of synapses per bouton 24 h after the induction of LTP. Further, we demonstrate that this increase is specific to mushroom spines and not attributable to their division. The present investigation thus fills the gap existing between behavioural and in vitro studies on the role of MSB formation in synaptic plasticity and cognitive abilities.

Alterations in synaptic curvature in the dentate gyrus following induction of long-term potentiation, long-term depression, and treatment with the N-methyl-D-aspartate receptor antagonist CPP.

Alterations in curvature of the post synaptic density (PSD) and apposition zone (AZ), are believed to play an important role in determining synaptic efficacy. In the present study we have examined curvature of PSDs and AZs 24 h following homosynaptic long-term potentiation (LTP), and heterosynaptic long-term depression (LTD) in vivo, in awake adult rats. High frequency stimulation (HFS) applied to the medial perforant path to the dentate gyrus induced LTP while HFS stimulation of the lateral perforant path induced LTD in the middle molecular layer of the dentate gyrus (DG). Curvature changes were analysed in this area using three dimensional (3-D) reconstructions of electron microscope images of ultrathin serial sections. Very large and significant changes in 3-D measurements of AZ and PSD curvature occurred 24 h following both LTP and LTD, with a flattening of the normal concavity of mushroom spine heads and a change to convexity for thin spines. An N-methyl-D-aspartate (NMDA) receptor antagonist CPP (3-[(R)-2-Carboxypiperazin-4-yl]-propyl-1-phosphonic acid) blocked the changes in curvature of mushroom and thin spine PSDs and apposition zones, actually increasing the concavity of mushroom spines as the spine engulfed the presynaptic bouton. In order to establish whether these changes resulted from the effect of the NMDA antagonist or from its coincidence with synaptic activation during testing we examined the effects of CPP alone on PSD and apposition zone curvature. It was found that CPP alone also caused a small decrease in curvature of both PSD and apposition zone of mushroom and thin 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.

Bilateral occlusion of carotid artery in awake hypertensive rats (SHR-SP) as a model of global cerebral ischemia.

Bilateral occlusion of carotid arteries in awake hypertensive rats (SHR-SP) was used as a model of global brain ischemia (duration of occlusion--until appearance of seizures). In normotensive rats (WKY), no seizures developed over 60 min. We revealed swelling of mitochondria in dendrites of hippocampal CA1 pyramidal cells, which was more pronounced in SHP-SP than in WKY rats. Blood pressure and heart rate in SHR-SP rats increased starting from the first minutes of occlusion, while in WKY rats these parameters remained unchanged. We proved that bilateral occlusion of the carotid arteries in awake SHR-SP rats can be used as an adequate model of global cerebral ischemia.

Reversible reduction in dendritic spines in CA1 of rat and ground squirrel subjected to hypothermia-normothermia in vivo: A three-dimensional electron microscope study.

A study was made at electron microscope level of changes in the three-dimensional (3-D) morphology of dendritic spines and postsynaptic densities (PSDs) in CA1 of the hippocampus in ground squirrels, taken either at low temperature during hibernation (brain temperature 2-4 degrees C), or after warming and recovery to the normothermic state (34 degrees C). In addition, the morphology of PSDs and spines was measured in a non-hibernating mammal, rat, subjected to cooling at 2 degrees C at which time core rectal temperature was 15 degrees C, and then after warming to normothermic conditions. Significant differences were found in the proportion of thin and stubby spines, and shaft synapses in CA1 for rats and ground squirrels for normothermia compared with cooling or hibernation. Hypothermia induced a decrease in the proportion of thin spines, and an increase in stubby and shaft spines, but no change in the proportion of mushroom spines. The changes in redistribution of these three categories of spines in ground squirrel are more prominent than in rat. There were no significant differences in synapse density determined for ground squirrels or rats at normal compared with low temperature. Measurement of spine and PSD volume (for mushroom and thin spines) also showed no significant differences between the two functional states in either rats or ground squirrels, nor were there any differences in distances between neighboring synapses. Spinules on dendritic shafts were notable qualitatively during hibernation, but absent in normothermia. These data show that hypothermia results in morphological changes which are essentially similar in both a hibernating and a non-hibernating animal.

Chronic restraint stress induces changes in synapse morphology in stratum lacunosum-moleculare CA1 rat hippocampus: a stereological and three-dimensional ultrastructural study.

Chronic restraint stress is known to affect the morphology and synaptic organization of the hippocampus, predominantly within CA3 but also in CA1 and dentate gyrus. In this study, we provide the first evidence for specific ultrastructural alterations affecting asymmetric axo-spinous synapses in CA1 stratum lacunosum-moleculare following chronic restraint stress (6 h/day, 21 days) in the rat. The structure of asymmetric axo-spinous post-synaptic densities was investigated using serial section three-dimensional reconstruction procedures in control (n=4) and chronic restraint stress (n=3) animals. Dendritic spine profiles (spine head+neck) associated with the sampled synaptic contacts (30 per animal) were also reconstructed in three-dimensions. Morphometric analyses revealed a significant increase in post-synaptic density surface area (+36%; P=0.03) and a highly significant increase in post-synaptic density volume (+79%; P=0.003) in the chronic restraint stress group. These changes were directly associated with 'non-macular' (perforated, complex and segmented) post-synaptic densities. A highly significant overall increase in the 'post-synaptic density surface area/spine surface area' ratio was also detected in the chronic restraint stress group (+27%; P=0.002). In contrast, no quantitative changes in spine parameters were found between groups. The Cavalieri method was used to assess the effects of chronic restraint stress exposure upon CA1 hippocampal volume. The mean volume of total dorsal anterior CA1 hippocampus was significantly lower in the chronic restraint stress group (-16%; P=0.036). However, when corrected for volume changes, no significant alteration in a relative estimate of the mean number of asymmetric axo-spinous synapses was detected in CA1 stratum lacunosum-moleculare between control and chronic restraint stress groups. The data indicate a structural remodeling of excitatory axo-spinous synaptic connectivity in rat CA1 stratum lacunosum-moleculare as a result of chronic restraint stress.

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 Ca(2+) 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.

Three-dimensional reconstruction of synapses and dendritic spines in the rat and ground squirrel hippocampus: new structural-functional paradigms for synaptic function.

Published data are reviewed along with our own data on synaptic plasticity and rearrangements of synaptic organelles in the central nervous system. Contemporary laser scanning and confocal microscopy techniques are discussed, along with the use of serial ultrathin sections for in vivo and in vitro studies of dendritic spines, including those addressing relationships between morphological changes and the efficiency of synaptic transmission, especially in conditions of the long-term potentiation model. Different categories of dendritic spines and postsynaptic densities are analyzed, as are the roles of filopodia in originating spines. The role of serial ultrathin sections for unbiased quantitative stereological analysis and three-dimensional reconstruction is assessed. The authors' data on the formation of more than two synapses on single mushroom spines on neurons in hippocampal field CA1 are discussed. Analysis of these data provides evidence for new paradigms in both the organization and functioning of synapses.

[Three-dimensional reconstruction of synapse and dendritic spines in the hippocampus of rats and ground squirrels: new paradigms of the structure and function of a synapse]. / Trekhmernye rekonstruktsii sinapsov i dendritnykh shipikov v gippokampe krys i suslikov: novye strukturno-funktsional'nye paradigmy raboty sinapsa.

The article reviews the literature data and results obtained by the authors concerning synaptic plasticity and remodeling of synaptic organelles in the central nervous system. Modern techniques of laser scanning confocal microscopy and serial thin sectioning for in vivo and in vitro studies of dendritic spines including a correlation between morphological changes and synaptic transmission efficiency are discussed, particularly, in relation to long-term potentiation. Organization of different types of dendritic spines and involvement of filopodia in spine genesis are examined. Significance of serial ultrathin sections for unbiased quantitative stereological analysis and three-dimensional reconstructions is discussed. The contact of one dendritic spine with two presynaptic boutons (multiple synapses) on both CA1 mushroom dendritic spines is discussed. The analyzed findings suggest new ideas for organization and functioning of synapses.

[Three-dimentional organization of synapses and astroglia in the hippocampus of rats and ground squirrels: new structural and functional paradigms of the synapse function]. / Trekhmernaia organizatsiia sinapsov i astroglii v gippokampe krys i suslikov: novye strukturno-funktsional'nye paradigmy raboty sinapsa.

The literature data and our own data on the synaptic plasticity and remodeling of synaptic organelles in the central nervous system are reviewed. Modern techniques of laser scanning confocal microscopy and serial thin sectioning for in vivo and in vitro studies of dendritic spines, including the relationship between morphological changes and the efficacy of synaptic transmission, are discussed using, in particular, a model of long-term potentiation. The organization of dendritic spines and postsynaptic densities of different categories as well as the role of filopodia in spine genesis were analyzed. It was shown that the method of serial ultrathin sections is the most effective for unbiased quantitative stereological analysis and 3D reconstructions. By using the refined method of serial ultrathin sections with subsequent three-dimensional reconstructions, the presence of giant mitochondria in hippocampal neuronal dendrites was demonstrated. It was shown that smooth endoplasmic reticulum forms a unified continuum with the outer membrane of the mitochondrial envelope within dendrites. It was suggested that this continuum provides calcium tunneling, which makes possible intracellular signal transduction during synaptic transmission. Evidence is presented indicating the presence of gap junctions ("electrical synapses") in the synapses of mammalian brain, as well as between glial processes, and between glial cells and neurons. Our data and the data of other authors show that glial cell processes form a structural and functional glial network, which modulates the functioning of the neuronal network. The connection of dendritic spines with the glial network is shown on 3D reconstructions by analyzing the neuropil volume in CA1 hippocampal area of ground squirrels in three functional states: normothermia, provoked arousal, and hibernation when brain temperature falls below 6 degrees C. The own data of the authors are discussed indicating the formation of more than five presynaptic boutons (multiple synapses) on both CA1 mushroom-like dendritic spines and CA3 thorny excrescences. On the basis of the analysis, new ideas of the organization and functioning of synapses were suggested.