Natalija Popović

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)

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.

Effect of physostigmine and verapamil on active avoidance in an experimental model of Alzheimer's disease.

The present study was performed to investigate and compare the effect of acetylcholinesterase inhibitor, physostigmine (0.045, 0.060 and 0.075 mg/kg sc, 30 min before the tests) and Ca-antagonist, verapamil (1.0, 2.5, 5.0 and 10.0 mg/kg sc, 30 min before the tests), on two-way active avoidance (AA) learning (acquisition and performance) in nucleus basalis magnocellularis (NBM)-lesioned rats. Bilateral electrolytic lesions of NBM induced significant decrease of acquisition and performance of AA responses in rats. Physostigmine (0.060 mg/kg) significantly improved only acquisition of AA, while verapamil (2.5 and 5.0 mg/kg) significantly improved both type of AA behavior in NBM-lesioned rats. These results suggest that altered calcium homeostasis might play significant role in pathogenesis of experimental induced Alzheimers disease (AD) and that administration of calcium antagonist such as verapamil might successfully ameliorate disturbances of learning and memory appeared after lesions of NBM.

Barnes maze performance of Octodon degus is gender dependent

Gender differences in spatial navigation have been widely reported in nocturnal rodent species. Here, for the first time we report gender differences in spatial learning and memory of Octodon degus, a long-lived diurnal hystricomorph rodent. In the present study, 16 months old male and female O. degus were tested in the 18-holes Barnes circular maze. The acquisition session consisted of four daily 4 min trials, during 10 days. Seven days later, the retention test was performed. To avoid the effect of hormonal fluctuation on spatial navigation, both the acquisition and the retention tests, were performed in 21-day regular cycling females in a period that corresponds to the diestrus phase of the estrus cycle. At the beginning of the acquisition, female degus were significantly slower than males to find the escape hole, but the situation reversed afterwards. Moreover, during the course of acquisition, females made significantly less reference memory errors, working memory errors as well as omission errors, than males. In both sexes, motivation and learning ceiling effects were reached at days 5-6 of the training. During the acquisition, females used more frequently a spatial strategy, while males preferably applied either serial, random or opposite strategies. The observed cognitive differences between male and female O. degus existed only during the acquisition period but not during the retention, indicating that acquisition and consolidation are differently influenced by gender.

Open Field Behavior in Nucleus Basalis Magnocellularis-Lesioned Rats Treated with Physostigmine and Verapamil

The present study was done to investigate and compare the effect of acetylcholinesterase inhibitor, physostigmine (0.030, 0.045, 0.060 and 0.075 mg/kg sc) and Ca-antagonist, verapamil (1.0, 2.5, 5.0 and 10.0 mg/kg sc) on open field behavior in male Wistar rats with bilateral electrolytic lesions of nucleus basalis magnocellularis (NBM). NBM-lesions produced a significant increase and decrease of ambulation and number of inner squares entered, and defecation, respectively, with no influence on grooming in rats exposed to novel environment. Physostigmine and verapamil in all tested doses, given 30 min before the test did not affect the open field behavior in control animals. In contrast to that, physostigmine (0.045, 0.060 and 0.075 mg/kg) and verapamil (2.5 and 5.0 mg/kg) significantly reduced ambulation and number of inner squares entered in NBM-lesioned rats. Also, physostigmine in a dose of 0.060 mg/kg significantly decreased defecation and in doses of 0.060 and 0.075 mg/kg the grooming, as well. On the other hand, verapamil only in a dose of 2.5 mg/kg significantly increased defecation. It could be concluded that lesions of NBM in rats induced disturbances in the open field behavior, which might be successfully ameliorate by physostigmine and verapamil treatment.

Time course of scopolamine effect on memory consolidation and forgetting in rats.

The effect of scopolamine on the consolidation and forgetting of emotional memory has not been completely elucidated yet. The aim of the present study was to investigate the time course of scopolamine effect on consolidation and forgetting of passive avoidance response. In a first experiment of the present study, we tested the effect of scopolamine (1mg/kg, i.p., immediately after acquisition), on 24h and 48h retention performance of the step-through passive avoidance task, in adult male Wistar rats. On the 24h retested trial, the latency of the passive avoidance response was significantly lower, while on the 48h retested trial it was significantly higher in scopolamine than in the saline-treated group. In a second experiment, we assessed the 24h time course of scopolamine (1mg/kg) effect on memory consolidation in passive avoidance task. We found that scopolamine administration only within the first six and half hours after acquisition improved memory consolidation in 48h retention performance. Finally, a third experiment was performed on the saline- and scopolamine-treated rats (given immediately after acquisition) that on the 48h retention test did not step through into the dark compartment during the cut-off time. These animals were retested weekly for up to first three months, and after that, every three months until the end of experiment (i.e., 15 months after acquisition). The passive avoidance response in the saline treated group lasted up to 6 weeks after acquisition, while in the scopolamine treated group 50% of animals conserved the initial level of passive avoidance response until the experiment end point. In conclusion, the present data suggest that (1) improving or impairment effect of scopolamine given in post-training periods depends on delay of retention trial, (2) memory consolidation process could be modify by scopolamine within first six and half hours after training and (3) scopolamine could delay forgetting of emotional memory.

Post-training scopolamine treatment induced maladaptive behavior in open field habituation task in rats.

The effects of scopolamine on memory consolidation are controversial and depend on several factors (i.e. site of administration, time of administration and testing, dose, cognitive task, experimental protocol, specie, strain, etc.). Generally, the range dose of systemic administered scopolamine, used in memory consolidation studies, has varied from 0.05 to 50 mg/kg. However, according to the literature, the most frequently used doses of scopolamine efficient on memory consolidation, are 1 and 30 mg/kg, low and high doses, respectively. In open field habituation studies only lower doses of scopolamine were used to test memory consolidation. Therefore, in the present study we compared the effects of low (1 mg/kg) and high (30 mg/kg) scopolamine dose, on the open field habituation task, in male Wistar rats. Scopolamine was administered immediately after the acquisition task and animals were retested 48 h later on. On the retested day, the ambulation and rearing in the open field decreased in the same manner in all tested groups. In saline- and 1 mg/kg scopolamine-treated animals, the time spent in grooming significantly decreased in the habituation task, while the same parameter significantly increased in animals treated with 30 mg/kg of scopolamine. The defecation rate significantly decreased (control group), maintained (1 mg/kg of scopolamine treated animals) or significantly increased (30 mg/kg of scopolamine treated group) on retention test. In conclusion, the present data suggest that post-training scopolamine administration does not affect locomotion neither exploration in the habituation to a novel environment, but increases defecation and grooming, two behaviours associated with fearful and stressful situations.

EFFECT OF NEURAL TRANSPLANTATION ON DEPRESSIVE BEHAVIOR IN RATS WITH LESIONED NUCLEUS BASALIS MAGNOCELLULARIS

Recent data of our group have shown that bilateral electrolytic lesions of the nucleus basalis magnocellularis (NBM) in rats reduced the escape behavior deficit that occurs in the learned helplessness test. The present study was done to establish the effect of intracerebral neural transplantation on the change in escape behavior of NBM-lesioned adult male Wistar rats in the learned helplessness test. At 2 days (NBM-ET) or 10 days (NBM-DT) after bi lateral electrolytic NBM-lesions, small fragments of fetal frontal cortex (18th day of gestation) were allotransplanted into the lesioned NBM. Ten days after neural transplantation, the learned helpless ness test was performed. The number of shocks that animals received before making an escape re sponse was significantly reduced in NBM-lesioned rats (p