Miroljub Popović

Importance of Immunological and Inflammatory Processes in the Pathogenesis and THERAPY of Alzheimer's Disease

The contribution of autoimmune processes or inflammatory components in the etiology and pathogenesis of Alzheimers disease (AD) has been suspected for many years. The presence of antigen-presenting, HLA-DR-positive and other immunoregulatory cells, components of complement, inflammatory cytokines and acute phase reactants have been established in tissue of AD neuropathology. Although these data do not confirm the immune response as a primary cause of AD, they indicate involvement of immune processes at least as a secondary or tertiary reaction to the preexisting pathogen and point out its driving-force role in AD pathogenesis. These processes may contribute to systemic immune response. Thus, experimental and clinical studies indicate impairments in both humoral and cellular immunity in an animal model of AD as well as in AD patients. On the other hand, anti-inflammatory drugs applied for the treatment of some chronic inflammatory diseases have been shown to reduce risk of AD in these patients. Therefore, it seems that anti-inflammatory drugs and other substances which can control the activity of immunocompetent cells and the level of endogenous immune response can be valuable in the treatment of AD patients.

Subfield and layer-specific depletion in calbindin-D28K, calretinin and parvalbumin immunoreactivity in the dentate gyrus of amyloid precursor protein/presenilin 1 transgenic mice.

The depletion of neuronal calcium binding proteins deprives neurons of the capacity to buffer high levels of intracellular Ca(2+) and this leaves them vulnerable to pathological processes, such as those present in Alzheimers disease (AD). The aim of the present study was to investigate the expression of the calcium binding proteins, calbindin-D28K, calretinin and parvalbumin in the dentate gyrus (DG) of amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic (Tg) mice and their non-Tg littermates, as well as the relation with the deposition of human amyloid beta (Abeta). We measured the expression of these three proteins at seven different rostro-caudal levels, and in the molecular, granular and polymorphic layers of the DG. We found that, except in the most caudal part of the DG, there is a substantial loss of calbindin-D28K immunoreactivity in all three layers of the DG in APP/PS1 mice compared with the non-Tg mice. Significant loss of calretinin immunoreactivity is present in most of the polymorphic layer of the DG of APP/PS1 mice compared with the non-Tg mice, as well as in the rostral and intermediate part of the inner molecular layer. Compared with the non-Tg mice parvalbumin immunoreactivity is significantly reduced throughout the whole polymorphic layer as well as in the rostral and intermediate part of the granular layer of DG in APP/PS1 mice. The relatively preservation of calbindin immunoreactivity in the caudal part of molecular and granular layers as well as calretinin immunoreactivity in the caudal part of polymorphic layer of the DG is likely related to the lower Abeta expression in those parts of DG. The present data suggest an involvement of calcium-dependent pathways in the pathogenesis of AD and indicate that there exists a subfield and layer-specific decrease in immunoreactivity which is related to the type of calcium-binding protein in APP/PS1 mice. Moreover, it seems that APP expression affects more the calbindin expression then parvalbumin and calretinin expression in the DG of APP/PS1 Tg mice.

Adult rat's offspring of alcoholic mothers are impaired on spatial learning and object recognition in the Can test

The aim of this study was to examine spatial and object recognition reference and working memory in adult offspring of Wistar rats exposed to ethanol in prenatal and/or preweaning period. For this purpose, four different conditions of the Can test were performed sequentially: spatial/object discrimination task, spatial orientation task, simple object recognition task and complex object recognition task. The results of present study shows: (1) the significant impairment in spatial learning and object recognition in animals exposed to alcohol during prenatal and/or preweaning period, (2) that cognitive dysfunction become increasingly evident with switching from simple to more sophisticated task, (3) that the most vulnerable period is the early neonatal period which corresponds to the third trimester gestational development in humans and (4) that during the developmental period, abrupt introduction or withdrawal of ethanol, rather than its continuous consumption, can produce higher cognitive deficit later on. In conclusion, moderate ethanol exposure during brain development produce long lasting impairment of spatial and recognition reference and working memory in adult rats offspring and these effects depend on the developmental period in which they were exposed to ethanol.

Neuroprotective effect of chronic verapamil treatment on cognitive and noncognitive deficits in an experimental Alzheimer's disease in rats

It is well known that disturbance of calcium homeostasis has a significant role in the development of neurodegenerative disorders, such as Alzheimers disease (AD). Our recent data suggest that acute treatment with the calcium antagonist verapamil can improve some behavioral deficits in an experimental model of AD. Therefore, the present study was done to establish the effect of chronically administered verapamil on cognitive and noncognitive behavior of rats with bilateral electrolitical lesions of nucleus basalis manocellularis (NBM)--an animal model of AD. The NBM lesions produce a deficit in performance of diverse behavior tests: active avoidance (AA), low level of fear (the open field test) as well as aggressive (the test of foot-shock induced aggression) and depressive (the learned helplessness test) behavior. Verapamil (1.0, 2.5, 5.0 and 10.0 mg/kg i.p.) or saline solution (1 ml/kg i.p.) were injected 24 hr after the lesion of NBM and then repeatedly administered during the next 8 days (twice a day). Performance of the two-way active avoidance test, the open field test, the foot shock-induced aggression test and the learned helplessness test were done on day 4 after the last verapamil or saline treatment (day 13 after the lesion). Verapamil in doses of 2.5 and 5.0 mg/kg significantly ameliorated the deficit in the performance of AA, the open field behavior, and the depression, but not the aggressive behavior. The obtained beneficial effect of chronic administered verapamil suggests that the regulation of calcium homeostasis during the early period after NBM lesions might be a reasonable way to prevent the behavioral deficits in an experimental model of AD.

Circadian System Functionality, Hippocampal Oxidative Stress, and Spatial Memory in the APPswe/PS1dE9 Transgenic Model of Alzheimer Disease: Effects of Melatonin or Ramelteon

Alzheimer disease (AD) is a neurodegenerative disorder that primarily causes β-amyloid accumulation in the brain, resulting in cognitive and behavioral deficits. AD patients, however, also suffer from severe circadian rhythm disruptions, and the underlying causes are still not fully known. Patients with AD show reduced systemic melatonin levels. This may contribute to their symptoms, since melatonin is an effective chronobiotic and antioxidant with neuroprotective properties. Here, the authors critically assessed the effects of long-term melatonin treatment on circadian system function, hippocampal oxidative stress, and spatial memory performance in the APPswe/PS1 double transgenic (Tg) mouse model of AD. To test if melatonin MT1/MT2 receptor activation, alone, was involved, the authors chronically treated some mice with the selective MT1/MT2 receptor agonist ramelteon. The results indicate that many of the circadian and behavioral parameters measured, including oxidative stress markers, were not significantly affected in these AD mice. During the day, though, Tg controls (Tg-CON) showed significantly higher mean activity and body temperature (BT) than wild-type (WT) mice. Overall, BT rhythm amplitude was significantly lower in Tg than in WT mice. Although melatonin treatment had no effect, ramelteon significantly reduced the amplitude of the BT rhythm in Tg mice. Towards the end of the experiment, Tg mice treated with ramelteon (Tg-RAM) showed significantly higher circadian rhythm fragmentation than Tg-CON and reduced circadian BT rhythm strength. The free-running period (τ) for the BT and locomotor activity (LA) rhythms of Tg-CON was <24 h. Whereas melatonin maintained τ at 24 h for BT and LA in both genotypes, ramelteon treatment had no effect. In the behavioral tests, the number of approaches and time spent exploring novel objects were significantly higher in Tg-CON than WT controls. Brain tissue analysis revealed significant reduction in hippocampal protein oxidation in Tg-MEL and Tg-RAM compared with Tg-CON animals. These results suggest that not all aspects of the circadian system are affected in the APPswe/PS1 mice. Therefore, care should be taken when extending the results obtained in Tg mice to develop new therapies in humans. This study also revealed the complexity in the therapeutic actions of melatonin and ramelteon in this mouse model of AD. (Author correspondence: jamadrid@um.es)

Multiple binge alcohol consumption during rat adolescence increases anxiety but does not impair retention in the passive avoidance task.

We investigated the effect of binge alcohol consumption on anxiety-related behavior and memory in adolescent male Wistar rats. Three consecutive daily sessions of ethanol administration (5 g/kg) were repeated weekly for 4 weeks. The retention of passive avoidance was measured weekly, 48 h following the treatment. Three days after the last memory test a novel object exploration test was done. There was no significant difference in step-through latency between the groups, but the ethanol-treated group displayed a significantly higher incidence of defecation, and an increased number of boluses during the passive avoidance test. The latency to explore a novel object was also higher, while the duration of exploration was significantly lower. Together, these data suggest that binge alcohol consumption in adolescent rats does not impair their memory in passive avoidance tasks, but may significantly increase their anxiety.

Behavioral and Adaptive Status in an Experimental Model of Alzheimer's Disease in Rats

Ten days after bilateral electrolytic lesions of nucleus basalis magnocellularis (NBM) we tested behavioral (spontaneous motor activity, acquisition and performance of two-way active avoidance, fear-response in open field test, foot shock induced aggression, depression-response in learned helplessness test) and adaptive status (body temperature at standard, hot and cold environment as well as cold restraint-induced gastric lesions) in adult male Wistar rats. Compared to intact control and sham-operated rats, the bilateral NBM-lesioned rats showed the significant impairment of learning behavior and reduced fear, aggression and depression as well as altered body temperature at standard and stressed conditions. Namely, it was established that body temperature in NBM-lesioned rats was significantly lower at standard laboratory conditions, but in these rats body temperature significantly was raised after exposing to cold and hot environment. On the other hand, spontaneous motor activity and number and length of cold restraint-induced gastric lesions (erosions and petechiae) in NBM-lesioned rats were similarly to those in both controls. It could be concluded that NBM plays a significant role in cognitive, emotional and adaptive processes in the rats.

Aging and time-of-day effects on anxiety in female Octodon degus

Animal models of anxiety have usually employed nocturnal species (e.g. rats and mice), and the tests used have been almost exclusively performed during the diurnal phase (lights on). Here, for the first time, we tested anxiety in a diurnal rodent, Octodon degus, according to its age (23 versus 40 months of age) and the time of the day. We employed three anxiety tests: object recognition, open field and light-dark tests, which were applied in the morning, at mid-day and in the late afternoon, respectively. Adult animals spent more time exploring a new object than aged animals. Nevertheless, there were no differences in the frequency of object exploration or in the latency to the first object approach between the groups. In the open field test, adult animals spent more time exploring (ambulation and rearing) than aged ones. Although both groups exhibited similar frequencies in transition from the dark to the light box in the light-dark test, adult animals spent significantly more time in the light, and expressed less anxiety when making the decision to cross over from the dark into the light area. In conclusion, there were no differences in anxiety between adult and aged animals in the morning session, although adult animals were more attentive when exploring a new object. However, in the mid-day and afternoon testing sessions, aged animals were more anxious than adults.

Long-term social isolation in the adulthood results in CA1 shrinkage and cognitive impairment.

Social isolation in adulthood is a psychosocial stressor that can result in endocrinological and behavioral alterations in different species. In rodents, controversial results have been obtained in fear conditioning after social isolation at adulthood, while neural substrates underlying these differences are largely unknown. Neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) are prominent modulators of synaptic plasticity underlying memory processes in many tasks, including fear conditioning. In this study, we used adult female Octodon degus to investigate the effects of long-term social isolation on contextual and cued fear conditioning, and the possible modulation of the synaptic levels of NCAM and PSA-NCAM in the hippocampus. After 6½ months of social isolation, adult female degus showed a normal auditory-cued fear memory, but a deficit in contextual fear memory, a hippocampal dependent task. Subsequently, we observed reduced hippocampal synaptic levels of PSA-NCAM in isolated compared to grouped-housed female degus. No significant differences were found between experimental groups in hippocampal levels of the three main isoforms of NCAM (NCAM180, NCAM140 and NCAM120). Interestingly, social isolation reduced the volume of the hippocampal CA1 subfield, without affecting the volume of the CA3 subregion or the total hippocampus. Moreover, attenuated body weight gain and reduced number of granulocytes were detected in isolated animals. Our findings indicate for the first time, that long-term social isolation of adult female animals induces a specific shrinkage of CA1 and a decrease in synaptic levels of PSA-NCAM in the hippocampus. These effects may be related to the deficit in contextual fear memory observed in isolated female degus.

The flavonoid apigenin delays forgetting of passive avoidance conditioning in rats

The present experiments were performed to study the effect of the flavonoid apigenin (20 mg/kg intraperitoneally (i.p.), 1 h before acquisition), on 24 h retention performance and forgetting of a step-through passive avoidance task, in young male Wistar rats. There were no differences between saline- and apigenin-treated groups in the 24 h retention trial. Furthermore, apigenin did not prevent the amnesia induced by scopolamine (1mg/kg, i.p., 30 min before the acquisition). The saline- and apigenin-treated rats that did not step through into the dark compartment during the cut-off time (540 s) were retested weekly for up to eight weeks. In the saline treated group, the first significant decline in passive avoidance response was observed at four weeks, and complete memory loss was found five weeks after the acquisition of the passive avoidance task. At the end of the experimental period, 60% of the animals treated with apigenin still did not step through. These data suggest that 1) apigenin delays the long-term forgetting but did not modulate the 24 h retention of fear memory and 2) the obtained beneficial effect of apigenin on the passive avoidance conditioning is mediated by mechanisms that do not implicate its action on the muscarinic cholinergic system.