Myelopeptides Reduce Morphine Tolerance in C57BL/6j Mice.

The development of morphine tolerance in C57BL/6j mice was estimated by the analgesic effect in tail-flick and hot plate tests. Morphine hydrochloride (10 mg/kg body weight) was administered to animals twice for 5 days and once on the sixth day, saline or myelopeptides were injected 15 min before morphine administration (2 µg/kg body weight). In the tail-flick test, all studied myelopeptides suppressed the development of tolerance to morphine and did not show their own analgesic activity. In the hot plate test, only three myelopeptides (MP2, MP5, and MP6) were found to reduce the formation of morphine tolerance. MP1 significantly reduced the analgesic effect of morphine on days 1-3 of administration, but contributed to the preservation of the analgesic effect during the period of tolerance development.

Effects of Specific Inhibitor of Phosphodiesterase 7 at the Late Stage of Long-Term Potentiation in Murine Hippocampal Slices.

Second messengers cAMP and cGMP play an important role in synaptic plasticity and memory consolidation. The inhibitors of phosphodiesterases, enzymes hydrolyzing these cyclic nucleotides, are actively studied as potential drugs for the treatment of various cognitive disorders and depression. We studied the effects of a new inhibitor of phosphodiesterase 7 AGF2.20 on the formation of long-term potentiation in hippocampal slices. Administration of AGF2.20 (10 nM) in 90 min after weak tetanization prevented a decrease in the amplitude of excitatory post-synaptic potentials and stabilized long-term potentiation. These data attest to the involvement of phosphodiesterase 7 in the development of synaptic plasticity in the hippocampus. The inhibitor AGF2.20 is considered for the further analysis as a promising substance for the treatment of cognitive impairments.

[Protective Effects of Myelopeptides in Neuroblastoma C-1300 Cell Culture].

The effects of bone marrow regulatory peptides--myelopeptides 1-6--in mouse neuroblastoma C-1300 were studied. Cultivation in presence of myelopeptides stimulated morphological differentiation of neuroblasts. Neuroprotective abilities of myelopeptides were shown on the models of morphine toxity and oxygen-glucose deprivation in neuroblastoma cell culture.

Age-specific peculiarities of formation of long-term posttetanic potentiation in OXYS rats.

OXYS rats with hereditary hyperproduction of active oxidative radicals and early disorders in the mitochondrial structure and functions are an interesting model for studies of age-specific features of synaptic plasticity. The formation of long-term posttetanic potentiation in the mossy fibers-CA3 pyramidal neuron system were studied in hippocampal slices from Wistar and OXYS rats aged 3 and 4.5 months (young), 11 (middle-aged), and 18 months (old). No appreciable age-related differences were detected in the amplitudes and latencies of stimulatory postsynaptic summary potentials of the mossy synapses evoked by test stimuli in Wistar and OXYS rat groups of different age and between the two strains. The capacity to induction and formation of long-term posttetanic potentiation and its value decreased in 18-month-old Wistar rats, which attested to disorders in synaptic plasticity of old animals. The capacity to induction and formation of long-term posttetanic potentiation and its value in OXYS were lower than Wistar rats of the same age in all the studied groups.

Participation of L-type voltage-gated calcium channels in facilitation of long-term potentiation during the formation of morphine dependence in rats.

Parameters of long-term potentiation in the system mossy fibers-CA3 pyramidal neurons in hippocampal slices in experimental animals vary during the formation of chronic opiate dependence. During the first day of morphine treatment, the value of potentiation was significantly lower than in controls. Starting from day 8 and at early stages of dependence (days 25-29), facilitation of long-term potentiation was recorded. Incubation of the slices with L-type Ca(2+)-channel blocker nifedipine did not change the response to the test stimuli and did not affect potentiation induction in hippocampal slices from control and morphine-treated animals. Nifedipine had no effect on long-term potentiation of mossy fibers in the control and at early terms of morphine treatment, but significantly reduced its facilitation at later terms.

[Polyhydroxylated steroid compounds from the Far Eastern starfish Distolasterias nipon].

Two new steroid glycosides: distolasteroside D6, (24S)-24-O-(beta-D-xylopyranosyl)-5alpha-cholestane-3beta,6alpha,8,15beta,16beta,24-hexaol, and distolasteroside D7. (22E,24R)-24-O-(beta-D-xylopyranosyl)-5alpha-cholest-22-ene-3beta,6alpha,8,15beta,24-pentaol were isolated along with the previously known distolasterosides D1, D2, and D3, echinasteroside C, and (25S)-5alpha-cholestane-3beta,4beta,6alpha,7alpha,8,15alpha,16beta,26-octaol from the Far Eastern starfish Distolasterias nipon. The structures of new compounds were elucidated by NMR spectroscopy and MALDI TOF mass spectrometry. Like neurotrophins, distolasterosides D1, D2, and D3 were shown to induce neuroblast differentiation in a mouse neuroblastoma C 1300 cell culture.

Neuroprotective effect of ultra-low doses of antibodies against S100 protein in neuroblastoma culture during oxygen and glucose deprivation.

Antibodies against S100 protein applied in high and ultra-high dilutions possess neuroprotective activity and maintain survival of neuroblastoma C-1300 cells under conditions of oxygen and glucose deprivation. The examined antibody preparations stimulated differentiation in neuroblastoma culture thereby demonstrating pronounced neurotrophic activity.

Neurotrophic effects of polyhydroxylated steroids and steroid glycosides in cultured neuroblastoma cells.

The effects of steroid compounds from Pacific Ocean starfishes were studied using cultured neuroblastoma C-1300 cells. Vital observations and examination of silver-impregnated preparations showed that the test substances in a concentration of 2-10 microM stimulate differentiation and improves survival of neuroblastoma cells under adverse conditions (similarly to neurotrophins). These substances in high concentrations (20-40 microM) had no effect or exhibited cytotoxic activity. The screening test allowed us to select several compounds for further studies of neurotrophic and neuroprotective properties.

Effect of antibodies to morphine in ultralow doses on induction of long-term potentiation in hippocampal slices from rats with chronic morphine dependence.

Antibodies to morphine produced after its chronic administration can contribute to changes in the central nervous system during opiate abuse. Facilitation of long-term posttetanic potentiation in mossy fibers of the hippocampus in rats with chronic morphine dependence can be reproduced in hippocampal slices from normal animals treated with antibodies to morphine. Incubation of hippocampal slices with ultralow doses of antibodies to morphine had no effect on control rats, but reduced facilitation of long-term potentiation in hippocampal slices from animals with chronic morphine dependence. This confirms the possibility of using ultralow doses of antibodies to morphine for therapeutic correction of mechanisms underlying the formation of drug abuse.

In vitro effects of bipathic treatment with antibodies in ultralow doses during long-term post-tetanic potentiation.

We studied the effects of individual or combination treatment with monoclonal antibodies 5F5-B6 in ultralow doses specifically marking mossy fibers in rat hippocampus and antibodies to S100 protein during long-term post-tetanic potentiation in hippocampal slices. The possible mechanisms of changes produced by therapeutic administration of antibodies in ultralow doses were revealed.

Effect of antibodies to morphine on synaptic plasticity of the hippocampus.

Incubation of hippocampal slices with antibodies to morphine did not change the total excitatory postsynaptic potential of mossy fibers, but markedly facilitated long-term posttetanic potentiation. Culturing of the organotypic hippocampal culture in the presence of 10 microM morphine increased the total excitatory postsynaptic potential of mossy fibers and reduced the probability of long-term posttetanic potentiation.

Membrane and synaptic effects of anti-S-100 are prevented by the same antibodies in low concentrations.

Effects of antiserum to S-100 protein (AS-100) in high (HC, corresponding to antiserum dilution 1:5 - 1:50) and low (LC, 1:10(12)) concentrations were studied in identified snail neurons and rat hippocampal slices. HC-AS-100 changed the frequency of action potential generation in spontaneously active neurons and blocked formation of long-term potentiation in mossy fiber synapses. LC-AS-100 alone did not affect any characteristic measured in our experiments, but 20 min pre-incubation of snail ganglia or hippocampal slices with LC-AS-100 abolished the effects of the same antibodies in HC. Simultaneous addition of both LC and HC did not prevent the development of HC-AS-100 effects. It seems possible that LC-AS-100 is capable of interaction with neuronal cells modifying their reaction to HC-AS-100.

Quantitative assay of S-100 protein in mouse brain cortex synaptosomes.

1. Data on the presence of S-100 protein in synaptic endings are revised, and evidence is given in favor of its localization inside mouse brain cortex synaptosomes and on the surface of their external membrane. 2. For identification of the S-100-specific polypeptide, proteins of external synaptosomal membranes were iodinated with lactoperoxidase fixed on cyanogen bromide (CNBr)-Sepharose, and after synaptosome lysis S-100-positive material was isolated by means of affinity chromatography antibodies to S-100 protein (a-S-100)-Sepharose. The molecular weight of the polypeptide obtained corresponded to that of S-100 subunits (10 kD), and iodine incorporation pointed to its localization on the surface of synaptosomal membranes. 3. With the help of antibodies labeled with horseradish peroxidase (a-S-100-HP) or 125I (a-S-100-125I), which do not penetrate into noninjured synaptosomes, the amount of S-100 protein on synaptosomal membranes was found to be 18.5 ng/mg total protein (as assayed with a-S-100-HP) or 95.33 ng/mg (as assayed with a-S-100-125I). 4. At the same time, the total S-100 protein content in synaptosomes measured by means of radioimmune analysis after their complete lysis turned out to be 284 +/- 0.84 ng/mg, i.e., a part of S-100 seemed to be inside synaptosomes. 5. Cosedimentation of water-soluble S-100 protein with the synaptosomal fraction during isolation was insignificant. Prefixation with glutaraldehyde or paraformaldehyde decreased the amount of material reacting with antibodies, possibly due to steric effects or denaturation of active centers. This could have influenced the earlier attempts to detect S-100 protein in synapses. Treatment of nonfixed synaptosomes with a conjugate of a-S-100 with colloidal gold made it possible to detect S-100-positive material on pre- and postsynaptic membranes, which confirms the biochemical data.

[An immunohistochemical analysis of rat hippocampal antigens in early postnatal ontogeny by using monoclonal antibodies]. / Immunogistokhimicheskii analiz antigenov gippokampa krysy v rannem postnatal'nom ontogeneze s ispol'zovaniem monoklonal'nykh antitel.

In order to study the molecular mechanisms of neurogenesis, monoclonal antibodies (MAbs) were produced against antigens of the developing rat hippocampus. MAb 3G7-F8 was used for immunohistochemical localization of the corresponding antigen of paraffin sections of the rat brain at days 0, 5, 14, and 21 of the postnatal development. In the hippocampus of newborn and 5-day-old rats, positive immunostaining was observed in the cytoplasm and proximal segments of processes of neurons located in granular, polymorph, and pyramidal layers, as well as in entorhinal cortex. In granule cell bodies and neurons of entorhinal cortex specific staining decreased by day 14 and disappeared by day 21 after birth, whereas neurons of pyramidal and polymorph layers remained immunopositive. Diffuse specific staining in the cerebellum was observed beginning from day 5 after birth in the Purkinje cell layer. On days 14-21 positive reaction was observed in Purkinje cell bodies and in the layer containing dendrites of Purkinje cells and parallel fibers. External and internal granular layers remained immunonegative. No specific staining was observed in other regions of the brain, as well as in the control slices. These data suggest that the antigen detected by the 3G7-F8 antibody is involved in the formation of the neuronal connections.

[Development of long-term post-tetanic potentiation and changes in S-100 protein contents in hippocampal slices of rats in different functional states]. / Razvitie dlitel'noi posttetanicheskoi potentsiatsii i izmenenie soderzhaniia belka S-100 v srezakh gippokampa krys s razlichnym funktsional'nym sostoianiem.

Male rats of the strains with low (LE) high excitability (HE) of the nervous system have been used in this study. Half of the animals of each strain were neurotized in accordance with the Hecht's scheme. In the hippocampal slices of the non-neurotized LE rats there was a significant increase of the populational spike amplitude during development of LTP as compared with the opposite group of the animals. The LTP formation in the LE strain of rats caused a decrease in the S-100 protein content in the water-soluble, and an increase in the membrane-bound fraction of the protein. Similar results we have observed with the non-inbred Wistar rats but not with the HE strain of the animals. The levels of the water-soluble S-100 protein fraction were also higher in the hippocampuses and entorenal cortices, but not in the cerebellae of the LE strain, as compared with the HE strain of the rats. No differences have been found in the membrane-bound fraction of S-100 protein.

[Monoclonal antibodies to individual antigens of the nervous system of the snail, Helix pomatia]. / Monoklonal'nye antitela k individual'nym antigenam nervnoi sistemy vinogradnoi ulitki Helix pomatia.

Seven positive hybridoma clones were chosen by immunoenzyme analysis amons 103 clones obtained by hybridization of NSO plasmocytoma cells and splenocytes from BALB/C mice, immunized with snail's nervous system antigens. Specific binding of Mabs with neuron cytoplasmic antigens was indicated on cryostat sections of visceral, pedal and cerebral ganglia. The Mabs obtained could be used for the study of physiological role of antigens identified.

Alterations in calmodulin and S-100 protein content of hippocampal slices during long-term potentiation.

The content of calmodulin and S-100 protein in fractions of rat hippocampal slices was assayed by solid phase radioimmunology and radial immunodiffusion, respectively. One hour after tetanization (electrical stimulation of area dentata granular cells and recording from CA3 pyramids) an inverse translocation of these Ca++-binding proteins was observed: an increase in the calmodulin content in the water-soluble and a decrease in the Lubrol-soluble fractions, while an increase in S-100 protein in the Triton-soluble and a decrease in the water-soluble fractions occurred. The results are suggestive of a regulatory function of these proteins in events during repetitive stimulation of a synaptic input. The calmodulin increase in the cytosolic compartment may reflect the involvement of Ca++-calmodulin dependent intraneuronal metabolic processes underlying the induction and/or temporary maintenance of neuronal functional changes occurring after repeated or intense synaptic activity. The elevated S-100 protein level in the membrane compartment might be interpreted in terms of functionally induced redistribution in that neuronal cells are provided with additional amounts of S-100 protein originating from the surrounding glial cells which store large amounts of soluble S-100 protein.