The role of histamine receptors in the consolidation of object recognition memory.

Findings have shown that histamine receptors in the hippocampus modulate the acquisition and extinction of fear motivated learning. In order to determine the role of hippocampal histaminergic receptors on recognition memory, adult male Wistar rats with indwelling infusion cannulae stereotaxically placed in the CA1 region of dorsal hippocampus were trained in an object recognition learning task involving exposure to two different stimulus objects in an enclosed environment. In the test session, one of the objects presented during training was replaced by a novel one. Recognition memory retention was assessed 24 h after training by comparing the time spent in exploration (sniffing and touching) of the known object with that of the novel one. When infused in the CA1 region immediately, 30, 120 or 360 min posttraining, the H1-receptor antagonist, pyrilamine, the H2-receptor antagonist, ranitidine, and the H3-receptor agonist, imetit, blocked long-term memory retention in a time dependent manner (30-120 min) without affecting general exploratory behavior, anxiety state or hippocampal function. Our data indicate that histaminergic system modulates consolidation of object recognition memory through H1, H2 and H3 receptors.

Early postnatal maternal deprivation in rats induces memory deficits in adult life that can be reversed by donepezil and galantamine.

Early postnatal maternal deprivation is known to cause long-lasting neurobiological effects. Here, we investigated whether some of the cognitive aspects of these deficits might be related to a disruption of the cholinergic system. Pregnant Wistar rats were individually housed and maintained on a 12:12h light/dark cycle with food and water freely available. The mothers were separated from their pups for 3h per day from postnatal day 1 (PND-1) to PND-10. To do that, the dams were moved to a different cage and the pups maintained in the original home cage, which was transferred to a different room kept at 32 degrees C. After they reached 120-150 days of age, maternal-deprived and non-deprived animals were either sacrificed for brain acetylcholinesterase measurement, or trained and tested in an object recognition task and in a social recognition task as described by Rossato et al. (2007) [Rossato, J.I., Bevilaqua, L. R.M., Myskiw, J.C., Medina, J.H., Izquierdo, I., Cammarota, M. 2007. On the role hippocampal synthesis in the consolidation and reconsolidation of object recognition memory. Learn. Mem. 14, 36-46] and Lévy et al. (2003) [Lévy, F., Melo. A.I., Galef. B.G. Jr., Madden, M., Fleming. A.S. 2003. Complete maternal deprivation affects social, but not spatial, learning in adult rats. Dev. Psychobiol. 43, 177-191], respectively. There was increased acetylcholinesterase activity in hippocampus and perirhinal cortex of the deprived animals. In addition, they showed a clear impairment in memory of the two recognition tasks measured 24h after training. Oral administration of the acetylcholinesterase inhibitors, donepezil or galantamine (1mg/kg) 30min before training reversed the memory impairments caused by maternal deprivation. The findings suggest that maternal deprivation affects memory processing at adulthood through a change in brain cholinergic systems.

Ovariectomy impairs spatial memory: prevention and reversal by a soy isoflavone diet.

Since a previous study has shown that ovariectomy impairs spatial memory, we, herein, investigate the influence of pre- and post-treatment with a soy diet on the effects elicited by ovariectomy on spatial memory. In the pre-treatment, 20-day-old female Wistar rats were first fed for 60 days on a standard diet with casein (control) or a soy diet. At 80 days of age, the animals were assigned to one of the following groups: sham (submitted to surgery without removal of ovaries) and ovariectomized. One week after surgery, the rats were submitted to behavioral testing. In the post-treatment, 80-day-old female rats were assigned to one of the following groups: sham and ovariectomized. One week after surgery, animals were fed for 30 days with the same diet described above. Then, rats were submitted to water maze testing. Pre-treatment for two months before ovariectomy with the soy diet effectively prevented the increase in latency in finding the platform on the fifth day of training in the ovariectomized group. Ovariectomized rats subjected to soy diet post-treatment reversed the increase in latency to find the platform in the ovariectomized group on the fifth day of training and, the decrease in the time spent in target quadrant, the increase in the time spent in opposite quadrant and the latency to cross the platform location. Results show that both pre- and post-treatment protected against the impairment of memory, caused by ovariectomy. Post-treatment reversed various parameters of memory reference, indicating that post-treatment was more efficient than pre-treatment. Based on these findings, we suggest that soy diet (rich in isoflavones) may represent a novel therapeutic strategy to prevent or to treat cognitive symptoms found in some menopausal women.

Posttraining activation of CB1 cannabinoid receptors in the CA1 region of the dorsal hippocampus impairs object recognition long-term memory.

Evidence indicates that brain endocannabinoids are involved in memory processing. However, the participation of CB1 and CB2 cannabinoid receptors in recognition memory has not been yet conclusively determined. Therefore, we evaluated the effect of the posttraining activation of hippocampal cannabinoid receptors on the consolidation of object recognition memory. Rats with infusion cannulae stereotaxically aimed to the CA1 region of the dorsal hippocampus were trained in an object recognition learning task involving exposure to two different stimulus objects. Memory retention was assessed at different times after training. In the test sessions, one of the objects presented during training was replaced by a novel one. When infused in the CA1 region immediately after training, the non-selective cannabinoid receptor agonist WIN-55,212-2 and the endocannabinoid membrane transporter inhibitor VDM-11 blocked long-term memory retention in a dose-dependent manner without affecting short-term memory, exploratory behavior, anxiety state or the functionality of the hippocampus. The amnesic effect of WIN-55,212-2 and VDM-11 was not due to state-dependency and was completely reversed by co-infusion of the CB1 receptor antagonist AM-251 and mimicked by the CB1 receptor agonist ACEA but not by the CB2 receptor agonists JWH-015 and palmitoylethanolamide. Our data indicate that activation of hippocampal CB1 receptors early after training hampers consolidation of object recognition memory.

The role of the entorhinal cortex in extinction: influences of aging.

The entorhinal cortex is perhaps the area of the brain in which neurofibrillary tangles and amyloid plaques are first detectable in old age with or without mild cognitive impairment, and very particularly in Alzheimer's disease. It plays a key role in memory formation, retrieval, and extinction, as part of circuits that include the hippocampus, the amygdaloid nucleus, and several regions of the neocortex, in particular of the prefrontal cortex. Lesions or biochemical impairments of the entorhinal cortex hinder extinction. Microinfusion experiments have shown that glutamate NMDA receptors, calcium and calmodulin-dependent protein kinase II, and protein synthesis in the entorhinal cortex are involved in and required for extinction. Aging also hinders extinction; it is possible that its effect may be in part mediated by the entorhinal cortex.

Hypermethioninemia increases cerebral acetylcholinesterase activity and impairs memory in rats.

In the present study we investigated the effect of chronic hypermethioninemia on rat performance in the Morris water maze task, as well as on acetylcholinesterase (AChE) activity in rat cerebral cortex. For chronic treatment, rats received subcutaneous injections of methionine (1.34-2.68 micromol/g of body weight), twice a day, from the 6th to the 28th day of age; control rats received the same volume of saline solution. Groups of rats were killed 3 h, 12 h or 30 days after the last injection of methionine to AChE assay and another group was left to recover until the 60th day of life to assess the effect of early methionine administration on reference and working spatial memory of rats. AChE activity was also determined after behavioral task. Results showed that chronic treatment with methionine did not alter reference memory when compared to saline-treated animals. In the working memory task, we observed a significant days effect with significant differences between control and methionine-treated animals. Chronic hypermethioninemia significantly increased AChE activity at 3 h, 12 h or 30 days after the last injection of methionine, as well as before or after behavioral test. The effect of acute hypermethioninemia on AChE was also evaluated. For acute treatment, 29-day-old rats received one single injection of methionine (2.68 micromol/g of body weight) or saline and were killed 1, 3 or 12 h later. Results showed that acute administration of methionine did not alter cerebral cortex AChE activity. Our findings suggest that chronic experimental hypermethioninemia caused cognitive dysfunction and an increase of AChE activity that might be related, at least in part, to the neurological problems presented by hypermethioninemic patients.

Supplementation with vitamins E plus C or soy isoflavones in ovariectomized rats: effect on the activities of Na(+), K (+)-ATPase and cholinesterases.

Since a previous study demonstrated that ovariectomized rats present an activation of Na(+), K(+)-ATPase and acetylcholinesterase (AChE) activities, in the present study we investigated the influence of vitamins E plus C or soy isoflavones on the effects elicited by ovariectomy on the activities of these enzyme in hippocampus of ovariectomized rats. We also determined the effect of the same compounds on the reduction of serum butyrylcholinesterase (BuChE) activity caused by ovariectomy. Female adult Wistar rats were assigned to one of the following groups: sham (submitted to surgery without removal of the ovaries) and ovariectomized. Seven days after surgery, animals were treated for 30 days with a single daily intraperitoneous injection of vitamins E (40 mg/kg) plus C (100 mg/kg) or saline (control). In another set of experiments, the rats were fed for 30 days on a special diet with soy protein or a standard diet with casein (control). Rats were sacrificed after treatments and the hippocampus was dissected and serum was separated. Data demonstrate that vitamins E plus C reversed the activation of Na(+), K(+)-ATPase and AChE in hippocampus of ovariectomized rats. Conversely, soy protein supplementation reversed the increase of AChE activity, but not of Na(+), K(+)-ATPase activity, caused by ovariectomized group. Neither treatment was able to reverse the reduction of serum BuChE activity. Furthermore, treatments with vitamins E plus C or soy were unable to reverse the decrease in estradiol levels caused by ovariectomy. Our findings show that the treatment with vitamins E plus C significantly reversed the effect of ovariectomy on hippocampal Na(+), K(+)-ATPase and AChE activities. However, a soy diet that was rich in isoflavones was able to reverse just the increase of AChE. Neither treatment altered the reduction in serum BuChE activity. Taken together, these vitamins and soy may have a protective role against the possible brain dysfunction observed in some menopause women. Vitamins E plus C and soy isoflavones may be a good alternative as a novel therapeutic strategy.

alpha-Tocopherol and ascorbic acid prevent memory deficits provoked by chronic hyperprolinemia in rats.

In the present study we investigated the action of alpha-tocopherol and ascorbic acid on the effects elicited by chronic hyperprolinemia on rat performance in the Morris water maze. Rats received subcutaneous injections of proline (experimental group) twice a day, with 10 h-interval, from the 6 to 28th days of age or an equivalent volume of 0.9% saline solution (controls). Half of the proline-treated group also received intraperitoneal administration of alpha-tocopherol (40 mg/kg) and of ascorbic acid (100 mg/kg) from the 6 to 28th days of life. On the 60th day of life, rats were subjected to testing in the water maze. Results show that chronic proline administration provokes impairment on spatial learning in reference memory task, as revealed by the increase of latency in acquisition, in the probe trial and in crossing over the platform location, as well as by the number of crossings, when compared to saline-treated animals. Proline-treated rats also demonstrated a reduced efficiency to find the platform position in the working memory task. Rats chronically treated with proline plus alpha-tocopherol and ascorbic acid had above effects prevented, suggesting the participation of oxidative stress in such effects. Our findings lend support to a novel therapeutic strategy, based on these vitamins, to the cognitive dysfunction associated with hyperprolinemia type II.

Vitamins E and C pretreatment prevents ovariectomy-induced memory deficits in water maze.

We investigated whether the pretreatment with vitamins E (alpha-tocopherol) and C (ascorbic acid) would act on ovariectomy-induced memory deficits in Morris water maze tasks. Adult female Wistar rats were divided into three groups: (1) naive (control), (2) sham (submitted to surgery without removal of ovaries) and (3) ovariectomized. Thirty days after surgery, they were trained in the Morris water maze in order to verify ovariectomy effects both on reference and working memory tasks. Results show that ovariectomized rats presented impairment in spatial navigation in the acquisition phase, as well as in the time spent in target quadrant and in the latency to cross over the location of the platform in test session, when compared to naive and sham groups (controls), in the reference memory task. Ovariectomy did not affect performance in the working memory task. Confirming our hypothesis, ovariectomized rats pretreated for 30 days with vitamins E and C had those impairments prevented. We conclude that ovariectomy significantly impairs spatial reference learning/memory and that pretreatment with vitamins E and C prevents such effect. Assuming this experimental memory impairment might mimic, at least in part, the cognitive deficit sometimes present in the human condition of lack of reproductive hormones, our findings lend support to a novel therapeutic strategy, based on vitamins E and C, to cognitive impairments in post-menopausal women.

Ovariectomy increases Na+, K+-ATPase, acetylcholinesterase and catalase in rat hippocampus.

In the present work we investigated the effect of ovariectomy on Na+, K+-ATPase and acetylcholinesterase (AChE) activities in rat hippocampus. We also studied some parameters of oxidative stress, namely total radical-trapping antioxidant potential (TRAP), thiobarbituric acid-reactive substances (TBA-RS), as well as the antioxidant enzyme activities superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. Our hypothesis is that ovariectomy might cause alterations in essential enzyme activities necessary to brain normal functioning and that these chances could be caused by oxidative stress. Female adult Wistar rats were divided into three groups: (1) naive (control); (2) sham-operated; and (3) ovariectomized. Thirty days after ovariectomy rats were sacrificed. Results showed that rats subjected to ovariectomy presented a significant increase in Na+, K+-ATPase, AChE and CAT activities, but did not change the oxidative stress parameters studied when compared to sham or naive rats. Since ovariectomy mimics postmenopausal changes, our findings showing alteration in the activities of brain Na+, K+-ATPase, AChE and CAT may be related to problems in postmenopausal women.

Ovariectomy enhances acetylcholinesterase activity but does not alter ganglioside content in cerebral cortex of female adult rats.

In the present work we investigated the effect of ovariectomy on acetylcholinesterase (AChE) activity and ganglioside content in cerebral cortex of female rats. We also studied the activity of butyrylcholinesterase (BuChE) in serum of these animals. Adult Wistar rats were divided into three groups: (1) naive females (control), (2) sham-operated females and (3) castrated females (ovariectomy). Thirty days after ovariectomy, rats were sacrificed by decapitation without anaesthesia. Blood was collected and the serum used for BuChE determination. Cerebral cortex was homogenized to determine AChE activity and extracted with chlorophorm:methanol for ganglioside evaluation. Results showed that rats subjected to ovariectomy presented a significant increase of AChE activity, but did not change the content and the profile of gangliosides in cerebral cortex when compared to sham or naive rats. BuChE activity was decreased in serum of rats ovariectomized. Our findings suggest that the alteration in the activity of brain AChE, as well as serum BuChE activity caused by ovariectomy may contribute to the impaired cognition and/or other neurological dysfunction found in post-menopausal women.

Metabolism of amino acids by cultured rat Sertoli cells.

Sertoli cells support spermatogenesis both spatially and energetically; for this reason, these cells have important adaptations. The energetic metabolism of Sertoli cells was adapted to provide lactate and pyruvate to developing germ cells, because these substrates are essential for spermatocytes and spermatids. In this study, we investigated whether Sertoli cells use alanine, leucine, valine, and glycine as energetic substrates and whether the simultaneous addition of other nutrients, such as glucose and glutamine, might affect the metabolism of these amino acids. Alanine, leucine, valine, and glutamine are almost totally oxidized to CO2 by these cells. In contrast, glycine has been demonstrated to be a poor energetic substrate, being mainly incorporated into proteins, and their metabolism did not change in the presence of palmitic acid, glucose, and/or glutamine. The metabolism of the 3 other amino acids was modified by palmitic acid; besides, glucose changed alanine, leucine, and valine oxidation. Glutamine decreased the oxidation of alanine, leucine, and valine to CO2. Conversely, both alanine and leucine decreased the oxidation of glutamine. Our present findings show that Sertoli cells can adapt its energy metabolism to the oxidative substrates available to fulfill their role in spermatogenic energetic supply.

In vivo and in vitro effects of homocysteine on Na+, K+-ATPase activity in parietal, prefrontal and cingulate cortex of young rats.

In the present study we determined the effect of chronic administration of homocysteine on Na+,K+-ATPase activity in synaptic membranes from parietal, prefrontal and cingulate cortex of young rats. We also studied the in vitro effect of homocysteine on this enzyme activity and on some oxidative stress parameters, namely thiobarbituric acid-reactive substances (TBA-RS) and total radical-trapping antioxidant potential (TRAP) in the same cerebral structures. For the in vivo studies, we induced elevated levels of homocysteine in blood (500 microM), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 micromol/g of body weight) twice a day at 8 h intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Rats were killed 12 h after the last injection. Chronic administration of homocysteine significantly decreased (50%) Na+,K+-ATPase activity in parietal, increased (36%) in prefrontal and did not alter in cingulate cortex of young rats. In vitro homocysteine decreased Na+,K+-ATPase activity and TRAP and increased TBA-RS in all cerebral structures studied. It is proposed that the alteration of Na+,K+-ATPase and induction of oxidative stress by homocysteine in cerebral cortex may be one of the mechanisms related to the neuronal dysfunction observed in human homocystinuria.