A. A. Yakovlev

Effects of tumor necrosis factor-alpha central administration on hippocampal damage in rat induced by amyloid beta-peptide (25-35).

Male Wistar rats received unilateral intrahippocampal injections of 3 nmol (3.18 μg) aggregated Aβ(25–35), intracerebroventricular bilateral injections of 0.5 μg human recombinant TNFα or both (Aβ(25–35) + TNFα‐treated animals). Seven days after the surgery brain sections were stained with cresyl violet (Nissl), for fragmented DNA (TUNEL), glial fibrillar acidic protein (GFAP) and isolectin B4‐reactive microglia. In addition, caspase‐3 activity in brain regions was measured fluorometrically. The morphology of the hippocampus after the injection of Aβ(25–35) or both Aβ(25–35) and TNFα (but not TNFα alone) showed cell loss in the CA1 pyramidal cell layer. The extension of neuronal degeneration measured in the CA1 field was significantly larger in Aβ(25–35)‐treated groups compared to the contralateral hemisphere of both vehicle‐treated controls and animals injected with TNFα alone. TNFα augmented the Aβ(25–35)‐induced damage, significantly increasing the extension of degenerating area. Administration of Aβ(25–35) caused reactive gliosis in the ipsilateral hemisphere as demonstrated by upregulation of GFAP expression and the presence of hypertrophic astrocytes in the hippocampus. This effect was much more prominent in the hippocampi of rats treated with Aβ(25–35) + TNFα but absent after administration of TNFα alone. In both Aβ(25–35)‐treated groups, the damaged area of the hippocampal CA1 field and lateral band of dentate gyrus displayed many darkly stained round isolectin B4‐positive phagocyte‐like microglial cells. Sparse TUNEL‐positive nuclei were found in the hippocampi of rats treated with Aβ(25–35) alone or together with TNFα, but not in the control brain sections or in brain sections of TNFα‐injected animals. The activity of caspase‐3 increased significantly in the ipsilateral hippocampus after the injection of Aβ(25–35). Surprisingly, administration of TNFα into the cerebral ventricles prevented this Aβ(25–35)‐induced increase in hippocampal caspase‐3 activity. The results are discussed from the perspective of dual (neuroprotective and neurodestructive) roles of TNF in the brain.

Amyloid-β (25-35) increases activity of neuronal NO-synthase in rat brain

Nitric oxide (NO) is a free radical with multiple functions in the nervous system. NO plays an important role in the mechanisms of neurodegenerative diseases including Alzheimers disease. The main source of NO in the brain is an enzymatic activity of nitric oxide synthase (NOS). The aim of the present study was to analyze the expression and activity of both neuronal (nNOS) and inducible (iNOS) isoenzymes in the cerebral cortex and hippocampus of rats after intracerebroventricular administration of amyloid-beta (A beta) peptide fragment A beta(25-35). NADPHd histochemistry as well as immunohistochemistry were also used to investigate nNOS and iNOS expression in rat brain. The data presented here show that A beta(25-35) did not influence levels of nNOS or iNOS mRNA or protein expression in both structures studied. A beta(25-35) activated nNOS in the cerebral cortex and hippocampus without effect on iNOS activity. A beta(25-35) decreased the number of NADPHd-expressing neurons in the neocortex, but it did not significantly influence the number NADPHd-positive cells in the hippocampus. The peptide had no effect on the number of nNOS containing cells. We hypothesize that increased synthesis of NO induced by A beta(25-35) is related to qualitative alterations of nNOS molecule, but not to changes in NOS protein expression.

Footshock stress alters early postnatal development of electrophysiological responses and caspase-3 activity in rat hippocampus

Postnatal changes in population spike (PS) amplitudes and caspase-3 activity were compared in the hippocampi of control rats and experimental animals subjected to a brief footshock on postnatal day (PD) 13. Footshock induced an increase in maximal PS amplitudes during the early period (from PD 14 to PD 16), however, the difference between stressed and control animals gradually decreased with age up to PD 21. No difference between hippocampal caspase-3 activity in control and footshock groups was revealed within the PD 14-17. However, caspase-3 activity in the latter group was significantly lower over the next period of postnatal development (PD 18-21). PS amplitudes in the slices of the footshock group significantly increased over PD 22-27. We suggest that footshock activates the development of hippocampal circuitry during early phases, this phenomenon mediating enhanced responsiveness as a result of an increased production of synaptic connections and related decrease in neuronal loss.

Electroconvulsive Shock Induces Neuron Death in the Mouse Hippocampus: Correlation of Neurodegeneration with Convulsive Activity

The relationship between convulsive activity evoked by repeated electric shocks and structural changes in the hippocampus of Balb/C mice was studied. Brains were fixed two and seven days after the completion of electric shocks, and sections were stained by the Nissl method and immunohistochemically for apoptotic nuclei (the TUNEL method). In addition, the activity of caspase-3, the key enzyme of apoptosis, was measured in brain areas immediately after completion of electric shocks. The number of neurons decreased significantly in field CA1 and the dentate fascia, but not in hippocampal field CA3. The numbers of cells in CA1 and CA3 were inversely correlated with the intensity of convulsions. Signs of apoptotic neuron death were not seen, while caspase-3 activity was significantly decreased in the hippocampus after electric shocks. These data support the notion that functional changes affect neurons after electric shock and deepen our understanding of this view, providing direct evidence that there are moderate (up to 10%) but significant levels of neuron death in defined areas of the hippocampus. Inverse correlations of the numbers of cells with the extent of convulsive activity suggest that the main cause of neuron death is convulsions evoked by electric shocks.

Pentylenetetrazol kindling in rats: Is neurodegeneration associated with manifestations of convulsive activity?

Structural changes in neurons and measures of oxidative stress were studied in the hippocampus of rats tolerant (ST) and sensitive (SS) to developing clonic-tonic seizures in conditions of pentylenetetrazol kindling. Sequences of 11 injections of pentylenetetrazol significantly decreased the number of normal neurons in hippocampal field CA1 in SS rats, this effect being seen in both hippocampal field CA1 and the dentate fascia in ST rats. Decreases in the numbers of normal neurons were accompanied by increases in the numbers of damaged cells in field CA4 in rats of both groups. After 21 injections, decreases in the numbers of normal neurons were seen in field CA1 in both SS and ST rats, while the numbers of damaged neurons were significantly greater than control only in ST rats in fields CA1 and CA4. The glutathione level was significantly lower in the hippocampus in both groups of rats than in controls. Thus, rats “ tolerant” to developing convulsions show signs of oxidative stress and neurodegenerative changes in the hippocampus. This suggests that oxidative neuron damage leading to neurodegeneration in the pentylenetetrazol kindling model is not directly associated with convulsive activity.

Crosslinkers and their utilization for studies of intermolecular interactions

We analyzed a new direction of studies of intermolecular interactions in cells with the use of crosslinkers and the possible utilization of crosslinkers in neurochemical studies. We reviewed current crosslinkers and described their main physicochemical characteristics. We also described the main specific features of utilization of crosslinkers in biological studies and, especially, in studies of the brain. We presented the general scheme of a typical experiment.

A secreted caspase-3-substrate-cleaving activity at low pH belongs to cathepsin B: a study on primary brain cell cultures

The cysteine proteases caspase-3 and cathepsins are involved in both neuronal plasticity and neuropathology. Using primary neuroglial and glial cerebellar cultures, the pH dependence of cleavage of a synthetic caspase-3 substrate, Ac-DEVD-AMC, was studied. At acidic pH, cathepsin B cleaved Ac-DEVD, this activity being significantly higher than that of caspase-3 at pH 7.4. This activity is blocked by peptide inhibitors of both caspase-3 and cathepsin B. Substitution of culture medium for balanced salt solution stimulated cathepsin B secretion in both types of cultures. Ischemia (oxygen-glucose deprivation) significantly decreased secretion of cathepsin B activities into the culture medium.

Inhibition of Caspase-3 Blocks Long-Term Potentiation in Hippocampal Slices

Incubation of rat hippocampal slices with the specific caspase-3 inhibitor Z-DEVD-FMK led to suppression of long-term potentiation (LTP), which developed over a period of time. Incubation of Z-DEVD-FMK for 4 h and more prevented tetanization from evoking LTP; Z-DEVD-FMK had no effect on baseline measures of synaptic plasticity and short-term plasticity (population spike amplitude, level of facilitation in conditions of paired stimuli). These data provide the first evidence for the involvement of a mechanism mediated by caspase-3 in the phenomenon of LTP.

Selective vulnerability of the hippocampus to interoceptive stress: Effects on interleukin-1β and erythropoietin

The selective vulnerability of the hippocampus to damaging extreme influences, ischemia, hypoxia, and metabolic stress is well known. However, the question of the selective vulnerability of the hippocampus to acute interoceptive stress remains to be investigated. Here, we studied the time course of the neuroinflammatory response to systemic administration of bacterial LPS at a high dose. We examined the level of proinflammatory cytokine interleukin-1β and the expression of the cytoprotective protein erythropoietin and correlations between these indices. We found that interoceptive stress induced a rapid inflammatory response in the nervous tissue, which may be considered as an adaptive response. The neuroinflammation declined in the neocortex but increased in the hippocampus. This effect may be considered as the deadaptation of this structure at this stage of the inflammatory response.

Content of mRNA for NMDA Glutamate Receptor Subunits in the Frontal Cortex and Striatum of Rats after Morphine Withdrawal Is Related to the Degree of Abstinence

We studied the expression of mRNA for genes, whose protein products regulate the glutamate/NO/cGMP signal cascade in the frontal cortex, striatum, midbrain, and hippocampus of rats with various degrees of spontaneous morphine withdrawal syndrome. The concentration of Grin2a mRNA (subunit of the NMDA glutamate receptor) in the frontal cortex increased mainly in animals with severe abstinence. By contrast, the expression of mRNA for the Grin2b subunit in the striatum decreased in animals with mild abstinence. Variations in the content of mRNA for other products of the cascade (isoforms of NO-dependent guanylate cyclase and cGMP-dependent protein kinase) after morphine withdrawal were not related to the degree of abstinence. Our results suggest that the region-specific expression of mRNA for certain subunits of the NMDA glutamate receptor after morphine withdrawal can determine the degree of abstinence.