The Role of NMDA Receptor Subunits in the Effect of Memantine on the Brain of Healthy Animals.

The non-competitive NMDA glutamate receptor antagonist memantine has neuroprotective properties and is the first non-cholinergic drug approved for the treatment of Alzheimer's disease. The purpose of this work was to test the hypothesis that injections of memantine to healthy animals can affect the subunit composition of NMDA receptors in the brain, which may explain the effects of its chronic administration. For this, the expression of subunits GluN1, GluN2A, GluN2B, and GluN2C was studied in the hippocampus and prefrontal cortex of rats after single or five subchronic injections of memantine. The results showed that the GluN2C subunit (GRIN2C) plays an important role in the effects of memantine; against the background of memantine treatment, the expression of this subunit markedly decreased in the prefrontal cortex, but not in the hippocampus, which significantly affected the excitation/inhibition balance in cortical structures.

Autophagy in Neurons of the Prefrontal Cortex and Hippocampus of Rats after Trimethyltin Chloride Intoxication.

Ultrastructural studies of the hippocampus and the prefrontal cortex of rats were performed 7, 30, and 50 days after their damage by neurotoxicant trimethyltin chloride (TMT). Significant damage to neurons was observed in both brain structures. In the hippocampus, a large number of autophagosomes (0.9±0.1 per µm2) appeared in the soma of neurons, dendrites, and axons in 7 days after intoxication. In addition, we observed the appearance of hyperchromic neurons with abnormal structure of mitochondria. In the prefrontal cortex, damaged neurons also contained autophagosomes, but their number was significantly lower (0.3±0.1 per µm2). The number of autophagosomes decreased with increasing the time after TMT administration: 30 days after injection, the content of autophagosomes in the hippocampus was 0.10±0.01 per µm2, while in the prefrontal cortex, autophagosomes were no longer found. We hypothesized that autophagy in the hippocampus was not effective enough to prevent neuronal death caused by the neurotoxicant.

Effect of Interleukin-10 on Localization of AMPA Receptors in Synapses during Long-Term Posttetanic Potentiation in Cultured Hippocampal Slices.

The effect of the anti-inflammatory cytokine IL-10 on the ultrastructural distribution of AMPA receptor GluR1 subunit in CA1 field of cultured hippocampal slices was studied by using immunohistochemical technique. It was found that long-term posttetanic potentiation increased the content of GluR1 in the postsynaptic density of the axo-spinous synapse. Addition of IL-10 in concentrations of 1 and 10 ng/ml to the medium facilitated long-term posttetanic potentiation thereby changing the distribution of GluR1 in the spine: the number of receptors increased in the cytoplasm and decreased in the postsynaptic density. It is assumed that activation of neuronal IL-10 receptors affects the distribution of AMPA receptors in axo-spinous synapses of hippocampal field CA1 through interplay of intracellular signaling pathways, thereby participating in the mechanisms of synaptic plasticity under normal conditions.

Effect of TGF-beta1 on long-term synaptic plasticity and distribution of AMPA receptors in the CA1 field of the hippocampus.

Using the methods of electrophysiology and immunohistochemistry, the effect of the transforming factor beta-1(TGF-ß1), an anti-inflammatory cytokine, on the long-term post-tetanic potentiation (LTP) in CA1 field hippocampal slices and the distribution of the GluR1 subunit of the AMPA receptor has been studied. It was shown that TGF-ß1 at a concentration of 10 ng/ml did not significantly affect the initial stage of LTP and substantially changed the distribution of synaptic AMPA receptors in response to tetanic stimulation. Twenty five minutes after the tetanization, the main pool of AMPA receptors (90%) was due to the postsynaptic density (PSD). By contrast, LTP in the presence of TGF-ß1 was accompanied by less pronounced changes in the distribution of AMPA receptors. Their localization in both pre- and postsynaptic regions remained nearly the same as that in the control. It may be suggested that the normal distribution of AMPA receptors in spinous synapses promotes the stabilization of potentiated synapses, thereby retaining LTP for longer terms.

Expression of mGlu Receptor Genes in the Hippocampus After Intoxication with Trimethyltin.

A variety of localization and signaling properties of eight subtypes of metabotropic glutamate receptors (mGluRs) in the brain provide glutamate an important regulatory role in many processes, including neurodegeneration and repair of neuronal damage. To identify specific subtypes of mGluRs, which are involved in neurodegeneration process, we assessed expression levels of their genes under pathophysiological conditions. Using quantitative real-time RT-PCR analysis, we studied transcription levels of mGlu2-5 and mGlu7 genes in the hippocampus after its damage by neurotoxicant trimethyltin chloride (TMT) in Wistar rats. This organotin compound is known to cause neurodegeneration in the brain, especially in the hippocampus. Morphological studies confirmed neuronal damage in CA3-CA4 subfields of the hippocampus 6 weeks after the treatment with TMT. Step-through passive avoidance test revealed memory deterioration in rat-treated TMT. Interestingly, 3 and 6 weeks after the treatment with TMT, expression levels of the mGlu2 and mGlu7 genes were not changed in comparison to the control values while expression level of mGlu4 genes was upregulated throughout the whole studied period of TMT action. The dynamics of mGlu3 gene expression revealed the existence of neuroinflammation 3 weeks after the treatment with TMT, which was further confirmed by the upregulation of cyclooxygenase-2 gene expression. The expression level of mGlu5 receptors was downregulated 6 weeks after the treatment with TMT. Our results revealed a significant role of mGlu4, mGlu5, and mGlu3 receptors in the neurodegenerative/reparative processes in the hippocampus after the treatment with TMT. Ligands of these receptor subtypes can be, therefore, considered potential therapeutic targets for prevention or reduction of neurodegeneration.

Effect of Pharmacological Modulation of Activity of Metabotropic Glutamate Receptors on Their Gene Expression after Excitotoxic Damage in Hippocampal Neurons.

Microinjection of kainic acid into rat hippocampus causes excitotoxic neuronal damage predominantly in the CA3 and CA1 fields. These lesions can be significantly reduced by simultaneous administration of MPEP, a negative allosteric modulator of type 5 metabotropic glutamate receptors, and LY354740, an agonist of type 2 metabotropic glutamate receptors. The decrease in neuronal death in the hippocampus during pharmacological modulation was paralleled by adaptive changes in gene expression. In the hippocampus, gene expression of type 5 postsynaptic metabotropic glutamate receptor was close to the control level, and in the frontal cortex expression of the gene of α1-subunit of the GABAA receptor returned to normal. In the frontal cortex, a reciprocal relationship was observed for type 2 metabotropic glutamate receptor: expression of the corresponding gene decreased in response to pharmacological activation.

Subacute activation of mGlu4 receptors causes the feedback inhibition of its gene expression in rat brain.

AIM: The present study aimed to understand the relationship between pharmacological activation of mGlu4 receptors and regulation of its gene in the hippocampus. MAIN METHODS: The expression level of the GRM4 gene, encoding mGluR4 receptors, was studied in the hippocampus and frontal cortex of rats after pharmacological activation of the receptor with positive allosteric modulator (E)-4-(2-Phenylethenyl)-2-pyrimidinamine (TCN 238). The drug was injected subcutaneously four times at a dose of 2mg/kg. The animals were previously trained with hippocampal-dependent task and after the treatment were tested for memory retrieval. The expression level of GRM4 was determined by qRT-PCR in control and experimental groups of animals one and five days post-treatment. KEY FINDINGS: We found that TCN 238 did not affect the performance of the learned task. However, the expression level of GRM4 in the hippocampus was reliable down-regulated five days after treatment with TCN 238. In addition, we showed that the expression level of GABRA1, encoding GABAA α-subunit was downregulated five days after the treatment in the frontal cortex. SIGNIFICANCE: Subacute pharmacological intervention in mGluR activity by the selective positive modulator TCN 238 has led to adaptive rearrangements of transcription processes in the hippocampus. Moreover, this regulation affected GABA system, confirming importance of the brain excitation-inhibition balance. Since the pharmacological influence on mGluR activity can be regarded as a promising tool aimed to correct brain dysfunction, the properties of mGluR modulators should be studied in more detail, including the level of gene transcription.

Expression of metabotropic glutamate receptors after hippocampal injury.

The expression of metabotropic glutamate receptors (mGluR2, mGluR3, mGluR4, and mGluR5) and dendritic cytoplasmic BC1 RNA in the hippocampus and frontal neocortex of Wistar rats was studied 1 and 4 weeks after intrahippocampal microinjection of kainic acid. The efficiency of glutamatergic transmission was shown to change not only in the injured hippocampus, but also in the neocortex. It was associated with variations in the expression of presynaptic (mGluR2, mGluR3, and mGluR4) and postsynaptic receptors (mGluR5).

Memory disorders in rats after impairment of the dorsal hippocampal CA3 field with kainic acid.

We studied the possibility of conditioning of food-procuring response in animals after impairment of the dorsal hippocampal region with kainic acid. Histological studies of brain sections showed that the greater part of dorsal hippocampal CA3 pyramidal neurons were lyzed in 2 weeks after kainic acid microinjection into the hippocampus. Morphological signs of necrosis were found in CA2 and CA4 neurons at the interface with CA3 field. Preinjection of anti-inflammatory cytokine IL-10 virtually did not prevent neuronal death. Damage to the hippocampus impaired learning of experimental animals and they required more attempts for reliable performance of the skill than the controls. Short-term memory of experimental rats was retained, which was proven by the results of single-day training. It was hypothesized that memory dysfunctions in animals after damage to the dorsal hippocampal CA3 field and to the hippocampus in general are determined by impaired transition of short-term into long-term memory.

[Metabotropic glutamate receptors as targets for new drug development].

The review is devoted to experimental investigations of metabotropic glutamate receptors and the properties of drugs (ligands) belonging to agonists, antagonists, and modulators of the activity of these receptors. Possibilities of the treatment of neurodegenerative disorders, cognitive disturbances in schizophrenia patients, and narcotic dependency by using drugs of this class are considered.

Expression of mitochondrial uncoupling protein UCP2 in the brain of rats after hippocampal injury inflicted by kainic acid.

The expression of mitochondrial protein UCP2 and cytochrome C-oxidase subunit III genes in the hippocampus and prefrontal cortex was evaluated by real-time PCR 3 and 7 days after microinjection of kainic acid into the dorsal hippocampus. In contrast to cytochrome C-oxidase subunit III mRNA, the level of UCP2 mRNA in the hippocampus increased 1 week after microinjection of kainic acid. The expression of both genes in the prefrontal cortex did not differ from the control. Presumably, activation of UCP2 synthesis in hippocampal injury indicates the neuroprotective effects of this protein.

Expression of mGluR5 and synaptophysin genes after injury to the dorsal Hippocampus, inflicted by cainic acid.

The expression of synaptophysin (vesicular protein) and mGluR5 (metabotropic glutamate receptor) genes was studied 3, 7, and 20 days after cainic acid injury of the dorsal hippocampal area in Wistar rats. The expression of both genes was characteristically reduced in the hippocampus. Twenty days after the exposure the expression of mGluR5 in this brain area reached the control level, while synaptophysin expression remained low. An opposite trend was observed in the frontal cortex: synaptophysin expression 20 days after exposure did not differ from the control, while mGluR5 expression was reduced. The peculiar time course of both genes' expression in the hippocampus and frontal cortex indicates the involvement of the frontal cortex in mechanisms of functional recovery after hippocampal injuries.

[Effect of intrahippocampal kainic acid on the behavior of rats and functional state of mitochondria in brain structures].

Cognitive processes and functional state of mitochondria in brain structures of Wistar rats were studied after intrahippocampal injection of kainic acid, an agonist of glutamate receptors. A single administration of 0.25 microg kainic acid into the dorsal part of the left and right hippocampi affected task retrieval and decreased inhibition of unrewarded responses. The injection of 0.75 microg kainic acid induced recurrent seizures and completely disorganized animal behavior. The functional state of mitochondria, as an important marker of excitotoxicity, was studied after intrahippocampal injections of kainic acid in the same doses. Kainic acid at 0.25 microg proved to activate the oxidative phosphorylation in hippocampal mitochondria. A higher (epileptogenic) dose of kainic acid inhibited mitochondrial respiration in the frontal cortex, but had an insignificant effect on mitochondrial respiration in the hippocampus. The disturbed interaction between the hippocampal system and frontal cortex after kainic acid administration can be the main factor of the revealed cognitive dysfunctions.

Energy metabolism in rat brain structures after injections of kainic acid into the frontal cortex.

We studied behavioral reactions of rats after injection of subconvulsive dose of kainic acid into the frontal cortex and mitochondrial respiration in the hippocampus and frontal and temporal cortex 17-20 days after administration of kainic acid. Retention of acquired habit and the dynamics of its extinction in experimental rats were close to those in the control group. Changes in mitochondrial function were observed only in the region of kainic acid injection: activation of phosphorylating respiration during oxidation of succinate. Presumably, the detected activation of energy metabolism in the frontal cortex indicates functional restructuring in mitochondria, aimed at compensation of disorders caused by the neurotoxin.

Effect of indomethacin on kainic acid-induced memory disorders.

Single administration of kainic acid to Wistar rats impaired quenching of the conditioned response and increased the number of perseverative reactions 2 weeks postinjection. Indomethacin prevented behavioral disturbances induced by kainic acid.

Studies of the reproduction of long-term memory during exposure to kainic Acid.

The effects of the neurotoxin kainic acid on a food-producing habit were studied in Wistar rats in an experimental chamber. Single doses of kainic acid at a subconvulsive dose (8 mg/kg, i.p.) were found to impair the habit, onset of impairment being delayed by several weeks rather than immediate. Conversely, administration of the neurotoxin at the convulsive dose (10 mg/kg) impaired reproduction of the habit with onset within several hours after treatment and persistence for periods of up to 10 days, though this dose did not prevent the acquisition of a new food-procuring habit. These defects in the reproduction of long-term memory traces were explained in terms of the characteristics of the effects of kainic acid on the hippocampal system, which is the most susceptible to systemic administration of the neurotoxin.

Influence of seizures on lipids of homogenate and neuronal and glial nuclei of rat neocortex.

Lipid composition of homogenate and neuronal and glial nuclei of the brain cortex of Wistar rats was studied under normal conditions and after seizures induced by injection of picrotoxin. Seizures increased contents of lysophosphatidylcholine, sphingomyelin, and total phospholipids in the homogenate. In neuronal nuclei contents of total phospholipids, sphingomyelin, phosphatidylcholine, and phosphatidylserine decreased, and contents of free fatty acids and lysophosphatidylcholine increased. In glial nuclei content of total phospholipids decreased and content of free fatty acids increased. The role of changes in the lipid composition of the neocortex cells during seizures and the involvement of lipid messengers in signal mechanisms are discussed.

[The long-term memory retrieval following kainic acid administration]. / Izuchenie vosproizvedeniia dolgovremennoi pamiati pri deistvii kainovoi kisloty.

Effect of the neurotoxin kainic acid to the food-procuring task were studied in Wistar rats. A single injection of the acid in subconvulsive dose (8 mg/kg) impaired the task performance within some weeks but not immediately after the treatment. Higher doses of kainic acid (10 mg/kg) impaired the task performance within a few hours after treatment for up to 10 days. The treatment did not prevent rat's learning of a new task in the same experimental chamber. The revealed deficit in the long-term memory retrieval might be explained by specific effects of kainic acid upon the hippocampal system.

Singlet-triplet splitting of geminate electron-hole pairs in conjugated polymers.

The singlet-triplet splitting of geminate polaron pairs in a ladder-type conjugated polymer has been studied by the thermally stimulated luminescence technique. The energy gap separating the singlet and triplet states of the geminate pairs is measured to be in the range of 3-6 meV, depending on the polymer morphology. The results of correlated quantum-chemical calculations on a long ladder-type oligomer are fully consistent with the observed values of the geminate polaron pair singlet-triplet gap. Such low splitting values have important implications for the spin-dependent exciton formation in conjugated polymers.

[Influence of experimental epileptogenesis on memory: the role of lipids in cognitive disorders]. / Vliianie éksperimental'nogo épileptogeneza na protsessy pamiati: rol' lipidov v mekhanizmakh kognitivnykh narushenii.

Learning and memory disorders accompanying epileptogenesis were studied in rats with the use of two experimental models of epilepsy, picrotoxin kindling and kainic treatment. Rise of exploratory activity and decrease in animal's capability for experimental extinction of a response were characteristic of the initial stage of epileptogenesis. It was suggested that a dysfunction of brain hippocampal system can be responsible for cognitive disorders. To reveal their mechanisms, lipid contents were determined in the neocortex and hippocampus in appropriate periods after exposure to epileptogenic factors. Long-term changes in hippocampal lipid spectrum were found five days after the exposure to kainic acid. In particular, after sodium valproate treatment (the compensation of kainic effects), the total content of phospholipids in hippocampus was decreased. The hippocampal sphingomyelin level dropped as a result of picrotoxin kindling. The sphingomyelin changes suggest some recovery processes in hippocampal cells and point to an adaptive role of membrane lipids in the mechanisms of the damaging epiptogenous effects.