M. V. Onufriev

Amyloid-β(25–35)-induced memory impairments correlate with cell loss in rat hippocampus

Amyloid beta-peptide (Abeta) plays an important role in the pathophysiology of Alzheimers disease. The relationship between amnesia induced by central administration of aggregated Abeta(25-35) and neurodegeneration in the hippocampus was investigated. One month after a single intracerebroventricular injection of Abeta(25-35) (15 nmol), male Wistar rats were tested in an eight-arm radial maze. A quantitative evaluation of cell number in hippocampal regions was carried out on H&E-stained brain sections of rats used in the behavioral study. Indices of free radical-mediated processes in the hippocampus were evaluated in additional groups of animals 1, 3, 5, and 30 days after surgery. Abeta(25-35) induced impairments of working and reference memory (RM) as well as neurodegeneration in the CA1 but not in the CA3 field of the hippocampus. A significant correlation between both reference and working memory (WM) impairments and the neuronal cell loss in the hippocampal CA1 region was demonstrated. A gradually developing oxidative stress was evident in the hippocampus of rats treated with Abeta(25-35) as indicated by the increase in 2-thiobarbituric acid (TBARS) reactive substances and superoxide generation. These data suggest the involvement of oxidative stress in Abeta(25-35)-induced neurodegeneration and a relation between memory impairment and neurodegeneration in the CA1 subfield of the hippocampus.

Single intracerebroventricular administration of amyloid-beta (25-35) peptide induces impairment in short-term rather than long-term memory in rats.

Ample experimental evidence indicates that intracerebral injection or infusion of amyloid-beta peptides (Abeta) to rodents induces learning and memory impairments as well as neurodegeneration in brain areas related to cognitive function. In the present study, we assessed the effects of a single intracerebroventricular (i.c.v.) injection of aggregated Abeta fragment (25-35) at a dose of 15nmol/rat on short-term and long-term memory in rats during the 6-month post-surgery period. The results demonstrate that Abeta(25-35)-induced memory impairments in spontaneous alternation behavior in a Y-maze at 17, 36, and 180 days after the surgery as well as in a social recognition task 110 days post-surgery. Abeta(25-35) also impaired spatial memory in an 8-arm radial maze, but did not influence performance of the step-down passive avoidance task. These results suggest that Abeta(25-35) preferably induces impairments of spatial and non-spatial short-term (working) memory rather than long-term memory in rats.

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.

Administration of aggregated beta-amyloid peptide (25-35) induces changes in long-term potentiation in the hippocampus in vivo.

Intracerebroventricular administration of aggregated β-amyloid protein fragment (25–35) (7.5 nmol/ventricle) was followed one month later by significant changes in the dynamics of long-term potentiation in the hippocampus in vivo, expressed as powerful and stable increases in the amplitude of evoked potentials. This phenomenon may be associated with oxidative stress in the hippocampus, which has previously been demonstrated in this model, and, thus, with disturbances in ion homeostasis.

Role of nitric oxide in the ethylcholine aziridinium model of delayed apoptotic neurodegeneration in vivo and in vitro.

The involvement of nitric oxide in neurodegenerative processes still remains incompletely characterized. Although nitric oxide has been reported to be an important mediator in neuronal degeneration in different models of cell death involving NMDA-receptor activation, increasing evidence for protective mechanisms has been obtained. In this study the role of nitric oxide was investigated in a model of NMDA-independent, delayed apoptotic cell death, induced by the neurotoxin ethylcholine aziridinium ethylcholine aziridinium both in vivo and in vitro. For the in vivo evaluation rats received bilateral intracerebroventricular injections of ethylcholine aziridinium (2nmol/ventricle) or vehicle. In the hippocampus a transient decrease in nitric oxide synthase activity occurred, reaching its lowest levels three days after ethylcholine aziridinium treatment (51.7+/-9.8% of controls). The decrease coincided with the maximal reduction in choline acetyltransferase activity as marker for the extent of cholinergic lesion. The effect of pharmacological inhibition of nitric oxide synthase was tested by application of various nitric oxide synthase inhibitors with different selectivity for the nitric oxide synthase-isoforms. Unspecific nitric oxide synthase inhibition resulted in a significant potentiation of the loss of choline acetyltransferase activity in the hippocampus measured seven days after ethylcholine aziridinium application, whereas the specific inhibition of neuronal or inducible nitric oxide synthase was ineffective. These pharmacological data are suggestive for a neuroprotective role of nitric oxide generated by endothelial nitric oxide synthase. In vitro experiments were performed using serum-free primary neuronal cell cultures from hippocampus, cortex and septum of E15-17 Wistar rat embryos. Ethylcholine aziridinium-application in a range of 5-80microM resulted in delayed apoptotic neurodegeneration with a maximum after three days as confirmed by morphological criteria, life-death assays and DNA laddering. Nitric oxide synthase activity in harvested cells decreased in a dose- and time-dependent manner. Nitric oxide production as determined by measurement of the accumulated metabolite nitrite in the medium was equally low in controls and in ethylcholine aziridinium treated cells (range 0.77-1.86microM nitrite). An expression of inducible nitric oxide synthase messenger RNA could not be detected by semiquantitative RT-PCR 13h after ethylcholine aziridinium application. The present data indicate that in a model of delayed apoptotic neurodegeneration as induced by ethylcholine aziridinium neuronal cell death in vitro and in vivo is independent of the cytotoxic potential of nitric oxide. This is confirmed by a decrease in nitric oxide synthase activity, absence of nitric oxide production and absence of inducible nitric oxide synthase expression. In contrast, evidence for a neuroprotective role of nitric oxide was obtained in vivo as indicated by the exaggeration of the cholinergic lesion after unspecific nitric oxide synthase inhibition by N-nitro-L-arginine methylester.

Periods of postnatal maturation of hippocampus: synaptic modifications and neuronal disconnection

The paired-pulse paradigm was used to study the maturation of CA1 population spikes (PS) in the hippocampal slices of Wistar rats. Measurements were taken daily, from postnatal day (PN) 14 to PN27. In the slices from younger animals, inputs exhibit strong paired-pulse profile, which may be associated with low synaptic efficacy. Both responses increased during the third week of life, however, PS1 increased faster so that the PS1/PS2 ratio increased during the early period and remained increased thereafter. This may reflect postnatal modifications of synaptic transmission mediating the increase in hippocampal responses. Modifications of synaptic efficacy are prevailing during early phases while other mechanisms take over at later stages. Partial correlation analysis suggests that the decline of PS amplitude after PN19 may be due to the decrease in the number of connected neurons rather than to modifications of the synaptic efficacy. Thus, the actual direction and magnitude of postnatal PS maturation is suggested to depend on the balance of these two factors. The transient decline of PS amplitude coincided with a period of caspase-3 activation. There was a clear general trend for caspase-3 activity to decrease before PN17, while the inverse trend was observed during next period up to PN21.

Caspase-like activity is essential for long-term synaptic plasticity in the terrestrial snail Helix.

Although caspase activity in the nervous system of mollusks has not been described before, we suggested that these cysteine proteases might be involved in the phenomena of neuroplasticity in mollusks. We directly measured caspase‐3 (DEVDase) activity in the Helix lucorum central nervous system (CNS) using a fluorometrical approach and showed that the caspase‐3‐like immunoreactivity is present in the central neurons of Helix. Western blots revealed the presence of caspase‐3‐immunoreactive proteins with a molecular mass of 29 kDa. Staurosporin application, routinely used to induce apoptosis in mammalian neurons through the activating cleavage of caspase‐3, did not result in the appearance of a smaller subunit corresponding to the active caspase in the snail. However, it did increase the enzyme activity in the snail CNS. This suggests differences in the regulation of caspase‐3 activity in mammals and snails. In the snail CNS, the caspase homolog seems to possess an active center without activating cleavage typical for mammals. In electrophysiological experiments with identified snail neurons, selective blockade of the caspase‐3 with the irreversible and cell‐permeable inhibitor of caspase‐3 N‐benzyloxycarbonyl‐Asp(OMe)‐Glu(OMe)‐Val‐Asp‐(OMe)‐fluoro‐methylketone prevented development of the long‐term stage of synaptic input sensitization, suggesting that caspase is necessary for normal synaptic plasticity in snails. The results of our study give the first direct evidence that the caspase‐3‐like activity is essential for long‐term plasticity in the invertebrate neurons. This activity is presumably involved in removing inhibitory constraints on the storage of long‐term memory.

A radiometric analysis of nitric oxide synthase activity in Hymenolepis diminuta

The free radical nitric oxide (NO) is a neuronal messenger which is synthesized from L-arginine and O2 by nitric oxide synthase (NOS). In the synthesis NO and L-citrulline are produced in a stoichiometric 1:1 relation. The activity of NOS was analysed in homogenates of the rat tapeworm Hymenolepis diminuta by measuring the formation of L-[3H]citrulline after incubation with L-[3H]arginine. The nature of NOS in H. diminuta was determined by studying the effect of 3 types of NOS inhibitors: (1) L-NAME, (2) EGTA, (3) 7-nitro-indazole. All inhibitors caused a significant but not complete reduction in the formation of L-[3H]citrulline. The results are discussed against the background of nerve cells and fibres positive for NADPH-diaphorase staining in H. diminuta.

NO synthase and free radical generation in brain regions of old rats : correlations with individual behaviour

WE have investigated the activity of NO synthase (NOS), and generation of free radicals (FRG) in selected brain regions of old male Wistar rats. Using the emotional resonance test, two groups of rats were selected for the experiment: passive, preferring dark space, and active, preferring light space. Highest NOS activity, and FRG were seen in cerebellum. As a rule, NOS activity was lower, and FRG higher in the respective brain regions of active rats than in passive rats. Positive linear inter-regional cross-correlations of NOS activity as well as of FRG were found. When all rats were assessed as one group, negative correlations between NOS, and FRG in cerebral cortex were revealed.