Palmitoylation of cdc42 Promotes Spine Stabilization and Rescues Spine Density Deficit in a Mouse Model of 22q11.2 Deletion Syndrome.

22q11.2 deletion syndrome (22q11DS) is associated with learning and cognitive dysfunctions and a high risk of developing schizophrenia. It has become increasingly clear that dendritic spine plasticity is tightly linked to cognition. Thus, understanding how genes involved in cognitive disorders affect synaptic networks is a major challenge of modern biology. Several studies have pointed to a spine density deficit in 22q11DS transgenic mice models. Using the LgDel mouse model, we first quantified spine deficit at different stages using electron microscopy. Next we performed repetitive confocal imaging over several days on hippocampal organotypic cultures of LgDel mice. We show no imbalanced ratio between daily spine formation and spine elimination, but a decreased spine life expectancy. We corrected this impaired spine stabilization process by overexpressing ZDHHC8 palmitoyltransferase, whose gene belongs to the LgDel microdeletion. Overexpression of one of its substrates, the cdc42 brain-specific variant, under a constitutively active form (cdc42-palm-CA) led to the same result. Finally, we could rescue spine density in vivo, in adult LgDel mice, by injecting pups with a vector expressing cdc42-palm-CA. This study reveals a new role of ZDHHC8-cdc42-palm molecular pathway in postsynaptic structural plasticity and provides new evidence in favor of the dysconnectivity hypothesis for schizophrenia.

[Spatial distribution of synaptic vesicles in CA1 hippocampal synapses under conditions of induced long-term potentiation in vitro].

Prolonged activation of excitatory glutamatergic synapses causes modifications in their functioning and ultrastructural organization. While postsynaptic activity-induced changes have been relatively well studied, the data on spatial dynamics of synaptic vesicles (SV) under conditions of synaptic activation are still lacking. Using organotypic hippocampal slice cultures as a model system and electron microscopy as a technique, we analyzed changes in SV numbers and their spatial distribution in spine synapses ofhippocampal CA1 area. Two approaches were used to activate synapses: a protocol of brief oxygen-glucose deprivation known to induce so-called anoxia-hypoglycemic long-term potentiation (LTP), as well as high frequency stimulation of Schaffer collaterals inducing LTP of evoked postsynaptic potentials in CA1 synapses. Observations during the first hour after stimulation (30 and 60 min time-points) have shown that in both cases active functioning of synapses leaded to decrease in the total SV number as well as to depletion of the readily releasable SV pool. Both experimental protocols caused a decrease in spatial clustering of SV which was more pronounced after anoxia-hypoglycemic LTP. Possible mechanisms and functional consequences of these phenomena are discussed.

Inhibition of T-type calcium channels protects neurons from delayed ischemia-induced damage.

Intracellular calcium increase is an early key event triggering ischemic neuronal cell damage. The role of T-type voltage-gated calcium channels in the neuronal response to ischemia, however, has never been studied. Using an in vitro model of ischemia-induced delayed cell death in rat organotypic hippocampal slice cultures, we show that T-type calcium channels inhibitors drastically reduce ischemic cell damage. Immunostaining studies reveal the existence of Ca(V)3.1 and Ca(V)3.2 types of low-voltage-activated calcium channels in rat organotypic hippocampal cultures. Low extracellular calcium (100 nM) or increase of intracellular calcium buffering ability by BAPTA-acetoxymethyl ester significantly reduced ischemia-induced neuronal damage. Pharmacological inhibition of the T-type calcium current by mibefradil, kurtoxin, nickel, zinc, and pimozide during the oxygen-glucose deprivation episode provided a significant protection against delayed neuronal death. Mibefradil and nickel exerted neuroprotective effects, not only if administrated during the oxygen-glucose deprivation episode but also in conditions of postischemic treatment. These data point to a role of T-type calcium currents in ischemia-induced, calcium-mediated neuronal cell damage and suggest a possible new pharmacological approach to stroke treatment.

[Structural organisation of CA1 zone in the hippocampus of rats in the experimental brain ischemia]. / Strukturna orhanizatsiia zony CA1 hipokampa shchuriv pry eksperymental'nii ishemiï mozku.

The dynamic of structural and ultrastructural changes of CA1 area of the hippocampus was examined in rats after 10 or 15 min of global ischemia (4 vessels occlusion, 4VO) followed by reperfusion. In the early period of reperfusion (15 min, 2 h), the structural changes in synaptic terminals were observed, without any significant signs of neuronal damage. These changes consisted in (i) the increase of the relative number of perforated and multiple synapses, and (ii) the synaptic vesicles rearrangement. Clear neuronal damage appeared morphologically at 24 h, and then developed for 3-6 days and resulted in the delayed damage and death of the hippocampal neurons.

Remodeling of synaptic membranes after induction of long-term potentiation.

Several morphological changes of synapses have been reported to be associated with the induction of long-term potentiation (LTP) in the CA1 hippocampus, including an transient increase in the proportion of synapses with perforated postsynaptic densities (PSDs) and a later occurrence of multiple spine boutons (MSBs) in which the two spines arise from the same dendrite. To investigate the functional significance of these modifications, we analyzed single sections and reconstructed 134 synapses labeled via activity using a calcium precipitation approach. Analyses of labeled spine profiles showed changes of the spine head area, PSD length, and proportion of spine profiles containing a coated vesicle that reflected variations in the relative proportion of different types of synapses. Three-dimensional reconstruction indicated that the increase of perforated spine profiles observed 30 min after LTP induction essentially resulted from synapses exhibiting segmented, completely partitioned PSDs. These synapses had spine head and PSD areas approximately three times larger than those of simple synapses. They contained coated vesicles in a much higher proportion than that of any other type of synapse and exhibited large spinules associated with the PSD. Also the MSBs with two spines arising from the same dendrite that were observed 1-2 hr after LTP induction included a spine that was smaller and a PSD that was smaller than those of simple synapses. These results support the idea that LTP induction is associated with an enhanced recycling of synaptic membrane and that this process could underlie the formation of synapses with segmented PSDs and eventually result in the formation of a new, immature spine.

Computer simulation approach to the quantification of immunogold labelling on plasma membrane of cultured neurons.

Cell culture is a convenient model system to study the expression of plasma membrane-bound proteins in nerve cells. Analysing it with an ultrastructural detail researchers often apply transmission electron microscopy together with immunogold labelling. Plasma membrane profiles are one-dimensional (1D) and provide little information about the topography of membrane-bound proteins. In order to convert 1D estimates of spatial arrangement for preembedding immunogold labelled proteins into two-dimensional (2D) quantities, namely the 2D pattern and density of labelling, this paper presents a simple computer simulation technique. This technique is based on a mathematical model permitting a simulated immunogold labelled membrane to be sampled in a way similar to microtome sectioning. An interlabel distance (ILD) estimate is used to define the position of immunogold particles in membrane profiles. In order to interpret experimental ILD measurements the simulated distribution best fit to the experimental data is selected and the corresponding 2D density and pattern of particle scattering are considered to explain the real situation. Various parameters including a cell section thickness, immunogold particle size etc can be adjusted to suit the demands of a particular experiment. The technique was applied to quantify the NCAM preembedding immunogold labelling in the plasma membrane of cultured rat hippocampal neurons.

Microglia in organotypic hippocampal slice culture and effects of hypoxia: ultrastructure and lipocortin-1 immunoreactivity.

Lipocortin-1 immunocytochemistry was used to study the various cell forms of microglia that appear during organotypic hippocampal tissue culture, as well as in the in vitro toxic hypoxia model. Antibodies against lipocortin-1 identified activated and phagocytic cells that were abundant in a slice after the plating of a culture: cells of the intermediate form at the later time-points of culturing, resting ramified microglia beginning from the seventh day of culturing, as well as activated and phagocytic cells that appeared in the slice after experimental toxic hypoxia induced by potassium cyanide treatment. Lipocortin-1-positive microglia cell forms corresponded well to the description of the microglia in vivo, and the morphology of microglia corresponded to the circumstances under which these cells were observed in slice cultures. Electron microscopic studies have demonstrated, for the first time, that microglia in organotypic slice culture preserve morphological features typical of different microglial forms in vivo, as well as specific contacts and interactions with the other neural tissue elements. After experimental toxic hypoxia, rapid changes in microglial ultrastructure and localization were observed, reminiscent of in vivo models of ischaemia. In conclusion, observations of microglial morphology and behaviour allow us to suggest that microglia in the organotypic culture preserve their essential characteristic features and properties, thus providing an important model system for studying the structure and function of these cells.

Microglia and astrocytes in the adult rat brain: comparative immunocytochemical analysis demonstrates the efficacy of lipocortin 1 immunoreactivity.

The distribution of glial cells (microglia and astrocytes) in different regions of normal adult rat brain was studied using immunohistochemical techniques and computer analysis. Lipocortin 1, phosphotyrosine, and lectin GSA B(4), were used for identification of microglia, while S100beta and glial fibrillary acidic protein identified astrocytes. Bioquant computerized image analysis was used to quantify and map the immunostained cells in sections from adult rat brain. If lipocortin 1 was used as a marker, more microglial cells were detected than with phosphotyrosine or lectin. The lipocortin 1-positive microglial population was most numerous (on average, 130+/-5 cells/mm(2) of the brain section area) in neostriatum, and least (51+/-4 cells/mm(2)) in cerebellum and medulla oblongata. In general, the density of lipocortin 1 microglia was higher in the forebrain, and lower in the midbrain, and the least in the brainstem and cerebellum. The number of S100beta astrocytes was two to three times larger than the number of microglial cells, and approximately two times greater than glial fibrillary acidic protein cells. A high density of astrocytes was found in the hypothalamus and hippocampus (more than 260 cells/mm(2)); they were more numerous in the white matter than in the gray matter. Fewer astrocytes were observed in the cerebral cortex, neostriatum, midbrain, medulla oblongata and cerebellum (less than 200 cells/mm(2)). Thus lipocortin 1 and S100beta were shown to be the most specific and reliable markers for microglia and astrocytes, respectively. The regional population differences demonstrated for lipocortin 1 microglia and S100beta astrocytes presumably reflect structural and functional specializations of the certain brain regions.

LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite.

Structural remodelling of synapses and formation of new synaptic contacts has been postulated as a possible mechanism underlying the late phase of long-term potentiation (LTP), a form of plasticity which is involved in learning and memory. Here we use electron microscopy to analyse the morphology of synapses activated by high-frequency stimulation and identified by accumulated calcium in dendritic spines. LTP induction resulted in a sequence of morphological changes consisting of a transient remodelling of the postsynaptic membrane followed by a marked increase in the proportion of axon terminals contacting two or more dendritic spines. Three-dimensional reconstruction revealed that these spines arose from the same dendrite. As pharmacological blockade of LTP prevented these morphological changes, we conclude that LTP is associated with the formation of new, mature and probably functional synapses contacting the same presynaptic terminal and thereby duplicating activated synapses.

[Endothelial cells during cultivation (a comparative analysis of methodological approaches)]. / Endotelial'ni klityny za umov kul'tyvuvannia (porivnial'nyi analiz metodychnykh pidkhodiv).

In the paper various methods for isolation and cultivation of endothelial cells are compared on the basis of the own data. The comparative analysis of the methods for isolation of endothelial cells from the vessel explant and by enzymatic treatment of aorta or coronary vessels is carried out. The methodical approaches to obtaining pure culture of endotheliocytes are being analysed.

[Changes in the topography of various proteins in cultured neurons after selective injury of various cytoskeletal elements with neurotoxins]. / Izmeneniia topografii nekotorykh belkov vneshnei membrany kul'tiviruemykh neironov pri izbiratel'nom povrezhdenii razlichnykh élementov tsitoskeleta neirotoksinami.

The spinal cord and hippocampal primary cultures were incubated with three neurotoxins (separately) known to impair the main components of the cytoplasmic cytoskeleton: 1) colchicine blocking the repolymerization of microtubules, 2) cytochalasin preventing elongation of actin filaments, and 3) beta, beta'-iminodipropionitrile (IDPN), causing disorganisation of neurofilaments. The distribution of surface membrane molecules on the surface of the neurons was evaluated in the ultrastructural study after treatment with the neurotoxins on the 5th, 12th, and 15th days in vitro (DIV). On the 12 DIV, the density of immunogold labelled neural cell adhesion molecules (NCAM) on IDPN-treated hippocampal neurons increased 1.45 times comparing to the controls. On the 5 DIV, the density of WGA (wheat germ agglutinin)-binding membrane glycoproteins increased 2.09 times on colchicine-treated neurons, and 3.98 times on cytochalasin-treated ones, whereas on the 12 DIV, the increase was 3.28 and 2.72 times, respectively, as compared to the control cultures of the same age. These data provide insights into the mechanisms of neurodegenerative changes in the nerve cells and into the relationship between the cytoskeletal elements and the surface molecules on the neuronal plasmatic membrane.

Androgen-sensitive period of humoral immune reactivity development in male mice.

The influence of androgens on the humoral immune reactivity development was investigated in male CBA mice. Positive correlation was found to exist between the plasma androgen level and the primary immune response throughout the pubertal period. The androgen-sensitive period of the humoral immune reactivity development coincided with the early stages of sexual maturation (from the 18th to the 30th day of life). In mice orchidectomized on the 18th day of life (18 ORx) the immune response followed the pattern of that in intact males up to 3 months of age but did not reach its peak at 4 months of age. On the contrary, in mice orchidectomized on the 30th day of life (30 ORx) the immune response was maximal at 2 months of age and remained high thereafter. Ten-day treatment of 18 ORx mice with daily injections of testosterone propionate at a dose of 0.1 or 1 mg/kg b.w. was ineffective in making their immune reactivity pattern similar to that in 30 ORx mice, while daily injections of 5 alpha-dihydrotestosterone (DHT, 1 mg/kg b.w.) was effective. The results suggest that the stimulating influence of testes on the humoral immune reactivity development in maturing mice is effected via DHT.

[The role of androgens in the regulation of the immune response in male CBA mice at various stages of ontogenesis]. / Rol' androgenov v reguliatsii immunnogo otveta u myshei-samtsov linii CBA na razlichnykh étapakh ontogeneza.

Gonadectomy of 18-day-old mice (the 1st group) causes retardation of the functional maturation of the immune system but that of 1 and 3-month-old mice (the 2nd group), on the contrary, intensifies the immune response. The treatment of the 1st group mice during 10 days with 5-alpha-DHT (100 micrograms/100 g b. w.) increases the immune response 1 month after the end of hormone therapy more than twice. The TP failed to evoke such effects. In the 2-nd group of mice TP as well as 5-alpha-DHT (10 micrograms, 100 micrograms) caused the same stimulatory effects and the TP (8 mg) and 5-alpha-DHT (3 mg)-decreased the immune response. The diverse effects of the TP and 5-alpha-DHT in immature and mature mice may be due to the age peculiarities of androgens metabolism.

[The immune response to heteroantigen of male mice at various stages of ontogeny and following gonadectomy]. / Immunnyi otvet na geteroantigen u myshei-samtsov na raznykh étapakh ontogeneza i posle gonadéktomii.

Experiments on male CBA mice have shown that androgens modulate the function of the immune system depending on its initial functional status. Studies of humoral immune response in mice during ontogeny have revealed that the most pronounced activation of the immune system coincides with the beginning of the sexual maturation (18-30 days). Gonadectomy of 18-days-old mice causes retardation of the functional maturation of the immune system but that of 1 month-old mice, on the contrary, intensifies the immune response. It is supposed that this period of life is one of the key periods of the humoral immunity development. Long-term decrease of the testosterone level in blood of mice castrated after puberty induces untimely extinction of the immune reactivity comparable with that in old animals.

[Testosterone content of the blood plasma in mature animals of various species during modeling of a pathological process in the testes using testicular antibodies]. / Soderzhanie testosterona v plazme krovi polozrelykh zhivotnykh raznykh vidov pri modelirovanii patologicheskogo protsessa v semennikakh s pomoshch'iu testikuliarnykh antitel.

Testosterone content in the blood plasma before and after administration of testicular antibodies (IgG and IgM) isolated from respective antisera was studied by a radioimmunoassay in 86 mature mice, rats and guinea-pigs. IgG at a dose of 0.4-2.4 mg of protein per 100 g of body mass decreased testosterone content in the blood plasma 1.6-4.2-fold early after administration (on the 3rd-6th day) in animals of all species. The level of testosterone remained below the basal values in the course of 33 days after the administration of immunoglobulins. A single administration of IgG at a dose of 2.4 mg of the protein resulted in a less damaging effect on the animals' testes than the same amount of the protein administered in fractions (at a dose of 0.8 mg three times a day). IgG produced a higher inhibiting effect than IgM administered at the same doses. Immunoglobulins of class G isolated from antisera which are specific to the testicular tissue, can be used for the simulation of a pathological process in the testes for a study of the etiology and pathogenesis of hypogonadism which is quite frequent in clinical practice.