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.

Partial kindling induces neurogenesis, activates astrocytes and alters synaptic morphology in the dentate gyrus of freely moving adult rats.

A partial kindling procedure was used to investigate the correlation between focal seizure development and changes in dendritic spine morphology, ongoing neurogenesis and reactive astrogliosis in the adult rat dentate gyrus (DG). The processes of neurogenesis and astrogliosis were investigated using markers for doublecortin (DCX), 5-bromo-2-deoxyuridine (BrdU) and glial fibrillary acidic protein (GFAP). Our data demonstrate that mild focal seizures induce a complex series of cellular events in the DG one day after cessation of partial rapid kindling stimulation consisting (in comparison to control animals that were electrode implanted but unkindled), firstly, of an increase in the number of postmitotic BrdU labeled cells, and secondly, an increase in the number of DCX labeled cells, mainly in subgranular zone. Ultrastructural changes were examined using qualitative electron microscope analysis and 3-D reconstructions of both dendritic spines and postsynaptic densities. Typical features of kindling in comparison to control tissue included translocation of mitochondria to the base of the dendritic spine stalks; a migration of multivesicular bodies into mushroom dendritic spines, and most notably formation of "giant" spinules originating from the head of the spines of DG neurons. These morphological alterations arise at seizure stages 2-3 (focal seizures) in the absence of signs of the severe generalized seizures that are generally recognized as potentially harmful for neuronal cells. We suggest that an increase in ongoing neurogenesis, reactive astrogliosis and dendritic spine reorganization in the DG is the crucial step in the chain of events leading to the progressive development of seizure susceptibility in hippocampal circuits.

[Photon radiation-induced structural and functional changes in the myocardium of hypertensive SHR rats].

Male rats were irradiated by a Korobkov photon light-emitting diode matrix with a maximum irradiation at 612 nm every day 1 h per day for 13 days. After a course of irradiation, the rhythmoinotropic characteristics of the cardiac muscle significantly improved. Exposure to photon radiation initiated an active rearrangement in myocytes as shown by a morphological analysis. Considerable changes were found in the structure of sarcoplasmic reticulum (SR); the area of SR profiles increased more than twofold compared to control. This suggests a proportional increase in the ability of SR to absorb calcium, due to both an increase in its buffer capacity and possibly, an improved functioning of Ca2+ ATPase of the reticulum. Probably, the photon therapy leads to the normalization of calcium homeostasis in myocytes and improvement of the characteristics of the cardiac muscle contraction-relaxation cycle. Furthermore, changes in the proportions of the myocardium capillaries (increased by 75% compared to control; p < 0.001) and the area of mitochondrial profiles of myocytes (increased by 13%; p < 0.05) were observed, which lead to more active metabolic processes and a rise in energy potential in myocardial cells after photon radiation treatment.

[Protective effect of hypothermia on brain neurons of rats exposed to ionizing radiation].

The conditions of the protein-synthesizing system in neurons of the hippocampus (areas CA1 and C A3) and of the cortex (sensomotor region) in rats subjected to y-irradiation at a dose of 8 Gy under hypothermia (16 - 18 degrees C) and hypoxia-hypercapnia were investigated by fluorescent and electron microscopy. Under hypothermia, the protein-synthesizing system was shown to be damaged to a lesser degree and to be restored faster in comparison with similar neurons in rats irradiated at room temperature. In rats irradiated under hypothermia, the rRNA biogenesis and the protein-synthesizing activity of polyribosomes were restored in two days. The protective influence of hypothermia did not spread to changes in membrane structures (endoplasmic reticulum and Golgy apparatus); i.e., a partial loss of integrity and possible transformation of their structure caused by the irradiation and the restoration of these structures occurred at a lower rate.

[Influence of ionizing radiation on the condition of the protein-synthesizing system in ground squirrel brain neurons at different functional states].

It was shown by fluorescent and electron microscopy that the physiological state of ground squirrels subjected to ionizing radiation at different phases of the torpor-normothermia cycle plays a determining role in the alteration of the conditions of the protein-synthesizing system in neurons of hippocampus fields CA1 and CA3 and sensomotor area of the brain. In ground squirrels irradiated under normothermia, the neurons were less radioresistant and restored slower compared with torpor animal cells, the distinctions being most expressed in CA1 field neurons. The effect of irradiation was minimum during the entrance into torpor and maximum during arousal. It was supposed that the inhibition of protein synthesis in the latter case occurred at the elongation stage when heavy polyribosomes were formed in neuron cytoplasm.

[The effect of hypothermia on the rat radioresistance].

The cooling of Wistar rats up to 15-19 degrees C under a condition hypoxia-hypercapnia increased the radioresistance with a dose reduction factor (DRF) of 1.4. To elucidate the mechanisms of hypothermia radioprotective effect was evaluated the functional state of rat neocortex using a electroencephalogram (EEG) as well as was studied the lipid composition of neocortex under the conditions of both normothermia and hypothermia. At 19-20 degrees C the activity within a wide range of frequencies in EEG was suppressed; the nonregular slow waves were recorded against a background of "silence". The reduction of EEG spectrum with increasing temperature began with the low frequencies. At 26-28 egresC the contribution of theta-rhythm (an indicator of brain activity level) in EEG reaches the normothermia value, from this point the rat brain starts to functionate as a whole system. At normothermia the similarity of neocortex lipid composition in nonhibernators (rats) and hibernators (ground squirrels) mammalians was noted. The difference is only in a higher content of phosphatidylinositol in rats. Rats falling into hypothermia state as well as ground squirrels into torpor is followed by a decrease of cholesterol content and the absence of significant changes of the phospholipid composition in neocortex tissues.

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.

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.

[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.