A. V. Revishchin

The neuronal insulin sensitizer dicholine succinate reduces stress-induced depressive traits and memory deficit: possible role of insulin-like growth factor 2

BackgroundA number of epidemiological studies have established a link between insulin resistance and the prevalence of depression. The occurrence of depression was found to precede the onset of diabetes and was hypothesized to be associated with inherited inter-related insufficiency of the peripheral and central insulin receptors. Recently, dicholine succinate, a sensitizer of the neuronal insulin receptor, was shown to stimulate insulin-dependent H2O2 production of the mitochondrial respiratory chain leading to an enhancement of insulin receptor autophosphorylation in neurons. As such, this mechanism can be a novel target for the elevation of insulin signaling.ResultsAdministration of DS (25 mg/kg/day, intraperitoneal) in CD1 mice for 7 days prior to the onset of stress procedure, diminished manifestations of anhedonia defined in a sucrose test and behavioral despair in the forced swim test. Treatment with dicholine succinate reduced the anxiety scores of stressed mice in the dark/light box paradigm, precluded stress-induced decreases of long-term contextual memory in the step-down avoidance test and hippocampal gene expression of IGF2.ConclusionsOur data suggest that dicholine succinate has an antidepressant-like effect, which might be mediated via the up-regulation of hippocampal expression of IGF2, and implicate the neuronal insulin receptor in the pathogenesis of stress-induced depressive syndrome.

Distribution of dopaminergic fibers and neurons in visual and auditory cortices of the harbor porpoise and pilot whale

The distribution of putative dopaminergic fibers in two sensory cortical areas in the brain of the harbor porpoise (Phocoena phocoena) and pilot whale (Globicephala melaena) was analyzed at the light and electron microscopic levels using tyrosine hydroxylase (TH) immunohistochemistry. The quantitative analysis of the distribution of labeled fibers demonstrates that the primary visual cortex located in the lateral gyrus and entolateral sulcus contains a denser dopaminergic innervation than the auditory cortex within the posterior portion of the presylvian gyrus. In both areas, TH-immunoreactive fibers are densest in layer I, while layers IIIab and VI have intermediate densities and layers II and IIIc-V have the lowest fiber counts. Layer I is characterized by the presence of very thick TH-immunoreactive fiber populations, in addition to the thin and varicose fiber plexus observed throughout the cortical layers. Electron microscopic analyses demonstrated that some of these thick fibers represent the dendrites of TH-immunoreactive neurons located in the deep portion of layer I. The patterns observed in the present study suggest that the dopaminergic projections to the neocortex in whales have a different organization than in terrestrial mammals, particularly rodents and primates. These differences may reflect the fact that during evolution, the cetacean neocortex has retained many of the cytoarchitectonic features that are usually observed only in proisocortical regions in progressive terrestrial mammals.

A New Population of Calretinin-Positive Cells, Presumptively Neurons, with Polymorphous Spines in the Mouse Forebrain

An immunohistochemical reaction was used to study the locations of calretinin-positive cells on frontal sections of the anterior part of the mouse cerebral cortex. A previously undescribed population of cells with a characteristic structure was found at the anterior horns of the lateral ventricles. These cells had small (8–10 μm) round bodies giving rise to one and occasionally two nodose processes bearing rare polymorphous spines (PS) and thickenings of irregular shape. The relatively thick primary processes branched into finer processes, which also formed thickenings and spines of different calibers and structures. Calretinin-positive cells with polymorphous spines (CR+PS) were located in the white subcortical matter, in layer VI, and, significantly more rarely, in layer V of the frontal area of the dorsomedial cortex close to the cingulum. In addition, CR+PS cells were present in the rostrodorsal part of the caudate nucleus-putamen complex, in the anterior olfactory nucleus, in the subependymal layer of the dorsolateral angle of the lateral ventricle and, more rarely, in its dorsal wall. In contrast to the situation in mice, CR+PS cells were not present in the brains of other animals (rats, rabbits, cats). CR+PS cells showed no colocalization of calretinin with GABA or other neuronal or glial markers. It is suggested that these cells represent a previously unknown, probably neuronal, type of cell in the mouse forebrain.

The influence of donor age, nerve growth factor, and cografting with drosophila cells on survival of peripherally grafted embryonic or fetal rat dorsal root ganglia.

Previous studies have shown that embryonic rat and human dorsal root ganglion (DRG) cells survive grafting to the cavity of extirpated adult rat DRG. Furthermore, grafted human embryonic neurons were shown to send axons peripherally and into the spinal cord, where they establish functional synaptic connections. This study analyzed the survival of orthotopically allografted rat DRG cells from embryonic stages 15 (E15) and 20 (E20), and the influence on their survival of nerve growth factor (NGF). NGF was delivered to the DRG transplants either by pump infusion or by cotransplantation of cells from Drosophila melanogaster, transgenic for human NGF. Lumbar DRGs of adult rats were removed and a collection of E15 or E20 DRGs placed in the cavity. One month after grafting the total number of DRG cells in the grafts was counted. Differentiation of subpopulations of DRG cells was estimated by counting cells immunostained for calcitonin gene-related peptide (CGRP), Griffonia simplicifolia agglutinin isolectin B4 (GSA), or heavy neurofilament protein (antibody RT97). The results show: i) similar survival of E15 and E20 grafts, with great variability in the survival of different subpopulations in E15 transplants, but a more consistent distribution of different phenotypes in E20 transplants; ii) infusion of NGF for 2 weeks increases the survival of E15 transplants, but has a negative effect on E20 transplants; iii) Drosophila cells transfected with human NGF gene survive peripheral xenografting and have a positive effect on the survival of the GSA- and CGRP-positive populations in E15 and E20 transplants; iv) Drosophila cells without the human NGF gene increase cell survival in E20 transplants. These data suggest that i) the effect of NGF is dependent on the embryonic stage of the transplants, ii) age-dependent sensitivity to NGF influences graft survival, and iii) transgenic Drosophila cells can be cotransplanted with embryonic neural tissue to the mammalian peripheral nervous system with a positive effect on the survival of neural grafts.

Evaluation of Antithrombotic Activity of Thrombin DNA Aptamers by a Murine Thrombosis Model

Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4–7.1 µmol/kg (14–70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

New calretinin-positive cells with polymorphous spines in the mouse forebrain during early postnatal ontogeny.

Immunohistochemical studies of calretinin (CR) in forebrain structures adjacent to the anterior horn of the lateral ventricle in adult mice allowed us to detect a population of previously unknown mono- and bipolar cells whose bodies and processes were coated with polymorphous spines (PS) (Morfologiya, 135, No. 3, 7–19 (2009)). CR-positive spiny (CR+PS) cells did not contain GAD67 and were located in the white matter and layers V–VI of the frontal area of the dorsomedial cortex close to the cingulum, the rostrodorsal part of the caudate-putamen, the anterior olfactory nucleus, and the subependyma of the dorsolateral angle of the lateral ventricle. We report here studies of the distribution of these cells in seven-day-old mice. Comparative topographic analysis of definitive and early CR+PS cells showed that in seven-day-old mice, CR+PS cells were absent from the sites at which they were seen in adults, i.e., the anterior olfactory cortex, the cortical plate, and the inner part of the neostriatum. In addition, small numbers of CR+PS-like cells were seen at this age within the dorsal migration pathway, at the anterior margin of the neostriatum, along the dorsal border of the neostriatum with the corpus callosum, in the subependymal layer of the lateral wall of the lateral ventricle, and in the cingulum area. These data demonstrate that CR+PS cells may have a postnatal origin. Experiments to verify this hypothesis were performed using postnatal administration of bromodeoxyuridine (BrdU) to mice aged 2–4 days, followed by assessment of brain sections fixed at age 20 days. Double immunolabeling of sections for CR and BrdU demonstrated the presence of CR+PS cells containing postnatally supplied BrdU. These data provide evidence that at least some CR+PS cells undergo mitosis at postnatal age. In all probability, during the period from 7 to 20 days of postnatal development, CR+PS cells migrate to the sites that they occupy in adult animals.

Neural Stem Cells in the Mammalian Brain

New fundamental results on stem cell biology have been obtained in the past 15 years. These results allow us to reinterpret the functioning of the cerebral tissue in health and disease. Proliferating stem cells have been found in the adult brain, which can be involved in postinjury repair and can replace dead cells under specific conditions. Numerous genomic mechanisms controlling stem cell proliferation and differentiation have been identified. The involvement of stem cells in the genesis of malignant tumors has been demonstrated. Neural stem cell tropism toward tumors has been shown. These findings suggest new lines of research on brain functioning and development. Stem cells can be used to develop radically new treatments of neurodegenerative and cancer diseases of the brain.

Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells.

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38°C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.

Transgenic cell cultures that synthesize neurotrophic factors and the possibility of therapeutic use of its cells

Under the leadership of Corresponding Member of the Russian Academy of Sciences L.I. Korochkin, the Laboratory of Neurogenetics and Developmental Genetics (Institute of Gene Biology, Russian Academy of Sciences) for many years has been conducting studies of nervous system development, neural cell differentiation, and application of gene and cell technology to cure neurodegenerative diseases. The results of the study initiated by L.I. Korochkin and continued by his scientific successors support the direction of allocation of transgenic neurotrofic factors and heat-shock proteins as neuroprotectors for cell therapy. Potential for usage of promoter of HSP70 heat-shock gene of Drosophila to create transgenic constructs for therapy has been shown. Further improvement of technology of nonvirus transfer for therapeutic genes, as well as production of multicomponent genetic constructs coding several therapeutic factors with synergy effect, would stimulate creation of efficient cell medicals to cure neurodegenerative diseases.

Hiding in the Shadows: CPOX Expression and 5-ALA Induced Fluorescence in Human Glioma Cells

Protoporphyrin IX (PpIX) is widely used in photodynamic diagnosis. To date, the details of molecular mechanisms underlying PpIX accumulation in malignant cells after 5-ALA administration remain unclear. The fluorescence of PpIX was studied in human glioma cells. Several cell cultures were established from glioma tumor tissue to study the differences between fluorescence-positive and fluorescence-negative human glioma tumors. The cell cultures demonstrated fluorescence profiles similar to those of source tumor tissues, which allows us to use these cultures in experimental research. Dynamics of the rates of synthesis and degradation of fluorescent protoporphyrin IX was studied in the cultures obtained. In addition, the expression of CPOX, an enzyme involved in PpIX synthesis, was evaluated. mRNA levels of heme biosynthesis enzymes were analyzed, and PpIX fluorescence proved to correlate with the CPOX protein level, whereas no such correlation was observed at the mRNA level. Fluorescence intensity decreased at low levels of the enzyme, which indicates its critical role in PpIX fluorescence. Finally, the fluorescence intensity proved to correlate with the proliferative activity.