In vivo evaluation of the hippocampal glutamate, GABA and the BDNF levels associated with spatial memory performance in a rodent model of neuropathic pain.

BACKGROUND: Patients with chronic pain usually suffer from learning and memory impairment which may significantly decrease their quality of life. Despite laboratory and clinical studies, the mechanism underlying this memory impairment remains elusive. We evaluated the effect of chronic pain on the glutamate and GABA levels and BDNF expression in the CA1 region of hippocampus as a possible explanation for memory impairment related to neuropathic pain. METHODS: In this respect, 30 male rats were randomly allocated to 3 groups as control, sham and neuropathic. Neuropathic pain was induced by a chronic constriction injury of the sciatic nerve (CCI) and mechanical allodynia and the spatial memory was assessed using the Von Frey filaments and Morris water maze respectively. To determine the potential mechanisms, the in vivo extracellular levels of glutamate and γ-aminobutyric acid (GABA) were measured by microdialysis and the brain-derived neurotrophic factor (BDNF) expression was determined by using western blots technique in the hippocampus on days 14 and 21 post-CCI. RESULTS: We showed that CCI impaired spatial learning and memory in Morris water maze (MWM) task. BDNF expression level and glutamate concentration significantly decreased in rats with chronic constriction injury of the sciatic nerve (P<0.001, F=7.3, F=23.23). In addition, GABA increased in hippocampal CA1 region (P<0.001, F=39.2) when the pain threshold was minimum. Nevertheless, these changes reversed while pain was relieved spontaneously. CONCLUSION: Chronic pain induced by constriction of the sciatic nerve impairs the spatial learning and memory function in rats. This effect exerts through the increase in GABA concentration and decrease in the glutamate and BDNF levels in the CA1 region of the hippocampus.

Memory-improving activity of Melissa officinalis extract in naïve and scopolamine-treated rats.

Melissa officinalis L. (Labiatae) traditionally used in treating neurological disorders has also been identified as a memory-enhancing herb. The extract of M. officinalis has a cholinergic property. The role of basal forebrain cholinergic neurons, the neurons that are destroyed in Alzheimer's disease (AD), in learning and memory, is also well known. The aim of this study is to investigate the role of cholinergic system on the memory improving activity of M. officinalis extract. The leaves of M. officinalis were extracted with ethanol 80% using the maceration method. Rats received intra-peritoneal injections of M. officinalis extract in different doses (50-400 mg/kg) alone or in combination with scopolamine (1 mg/kg) before being trained in a Morris water maze (MWM) in a single-day training protocol. After training, the acetylcholinesterase enzyme (AChE) activity was measured in the hippocampus. Administration of M. officinalis extract (200 mg/kg) could significantly enhance learning and memory of naïve rats (p<0.001) and significantly ameliorate scopolamine-induced learning deficit, but the effect of the extract was not dose dependent, and doses above 200 mg/kg could neither enhance memory in naïve rats nor reverse scopolamine-induced memory impairment. Also, inhibition of AChE activity was observed in both naïve and scopolamine-induced memory-impaired rats. These results suggest that M. officinalis can improve memory and that the cholinergic property of the extract may contribute to the memory-improving effects observed in this study. Then M. officinalis extract has potential therapeutic value in alleviating certain memory impairment observed in AD.

Effects of training in the Morris water maze on the spatial learning acquisition and VAChT expression in male rats.

BACKGROUND AND THE PURPOSE OF THE STUDY: It has been well established that cholinergic pathway plays an important role in learning and memory processes. The present study was designed to evaluate the effects of Morris water maze (MWM) training on spatial memory acquisition and expression of the vesicular acetylcholine transporter (VAChT) in male rats. METHODS: In this study, training trials of all groups of animals were conducted in the MWM task. Rats received one training session consisting of four trials per day which continued for another four consecutive days. Controls received visible platform MWM training. The escape latency, the traveled distance and swimming speed for each rat were recorded and used to evaluate the performance of the animal during training period. For evaluation of expression of VAChT protein levels, brain tissues from animals in each experiment were obtained immediately after the last trial on the related experimental day and processed for immunohistochemistry staining and western blotting analysis. RESULTS: There was a significant difference between animals subjected to one day training and those receiving four days of training in escape latency and travel distance. There were an apparent increase in VAChT immunoreactivity in the medial septal area (MSA) and CA1 region of the hippocampus in one day and four day trained animals compared with controls (visible group). Quantitative immunostaining analysis by optical density measurements in the CA1 region and evaluation of immunopositive neurons in medial septal area of brain sections confirmed qualitative findings. Assessment of VAChT protein level expression in hippocampus by western blotting evaluation showed the same pattern of immunohistochemistry results. CONCLUSION: Overall, results of this study reveal changes in cholinergic neuron activity in different stages of training in the MWM task. Data suggest that there is a significant level of cholinergic neuronal activity during early stages of the training especially in the hippocampus region that may contribute to the apparent increase in VAChT expression.

Formalin pain increases the concentration of serotonin and its 5-hydroxyindoleacetic acid metabolite in the CA1 region of hippocampus.

BACKGROUND AND THE PURPOSE OF THE STUDY: The hippocampal formation is involved in nociception. Prenatal serotonin depletion results in a significant decrease in the concentration of nociceptive sensitivity during the second phase of behavioral response in the formalin test. METHODS: A microdialysis probe was inserted via a guide cannula into the right CA1 region of the hippocampus. Extracellular serotonin (5HT) and its 5- hydroxyindoleacetic acid (5HIAA) metabolite overflow were collected every 10 min during the formalin test and measured by HPLC with electrochemichal detector. RESULTS: Compared to the sham group, formalin injection in the hind paw of the rat significantly increased 5HT after 10, 30, 40, and 50 min and increased 5HIAA after 10, 30, 40, 50, and 60 min collection time periods in hippocampal dialysate. (n=6 for each group at each sampling time). In the formalin treated rats serotonin and 5HIAA concentrations increased in the biphasic pattern in concert with the first and second phases of formalin pain. CONCLUSION: The hippocampal formation might be involved in the processing of nociceptive information and serotonin-related mechanisms in the hippocampus may play a role in the biphasic behavioral responses to formalin noxious stimulation.

The effect of severe zinc deficiency and zinc supplement on spatial learning and memory.

Zinc deficiency during pregnancy and during lactation has been shown to impair cognitive function and motor activity in offspring rats. In the present study, the effect of zinc deficiency and zinc supplement on spatial learning and memory in Morris Water Maze (MWM) and motor activity in open field were investigated. Pregnant rats after mating were divided to three groups. Control group fed a standard diet and a zinc deficient (ZnD) group fed a diet deficient in zinc (0.5-1.5 ppm) and a zinc supplement (ZnS) group fed a standard diet and enhanced zinc in the drinking water (10 ppm). All the diets were exposed during the last trisemester of pregnancy and during lactation. Rat's offspring in these groups were tested for spatial learning and memory in MWM at post natal day (PND) 56 and were tested for motor activity in open field at PND 66.The Escape Latency (EL) and Traveled Distance (TD) in the ZnD group were increased but Percentage of Time Spent in the target quadrant (PTS) was decreased compared to the control group. In addition, these were no significant differences in EL and TD, but PTS had significant increase in ZnS compared to the control group. In the open field, Total Distance Moved (TDM) and Time of Motor Activity (TMA) for the ZnD were decreased compared to the control group, but there were no significant differences in TDM and TMA between control and ZnS groups. These findings suggest that zinc deficiency during the last trimester of pregnancy and during lactation impaired spatial learning and memory in their offsprings and has also negative effect on motor activity. In addition, ZnS has a significant effect on spatial learning and memory but no effect on motor activity in their offsprings.

Role of the ventromedial hypothalamic orexin-1 receptors in regulation of gastric Acid secretion in conscious rats.

Orexins play an important role on the central nervous system to modulate gastric acid secretion. The orexin receptors are distributed within the hypothalamus, and expression of orexin-1 receptors (OX1R) is greatest in the anterior hypothalamus and ventromedial nucleus. Therefore, we hypothesised that ventromedial hypothalamic OX1R may be involved in the control of gastric acid secretion. To address this question, we examined the effects of orexin-A and a selective OX1R antagonist, SB-3345867, on gastric acid secretion in pyloric-ligated conscious rats. Intraventromedial injection of orexin-A (0.5-2 microg/microl) stimulated gastric acid secretion in a dose-dependent manner. This stimulatory effect of orexin-A persisted over 3 h. In some experiments, SB-3345867 (10 mg/kg i.p.) was administered 30 min before orexin-A or saline injections. We found that i.p. injection of SB-334867 suppressed stimulated gastric acid secretion induced by orexin-A (2 microg/microl). Atropine (5 mg/kg) also inhibited the stimulatory effect of central injection of orexin-A on acid secretion. In conclusion, the present study suggests that endogenous orexin-A acts on the ventromedial hypothalamus to stimulates acid secretion. This stimulatory effect is probably mediated through OX1R.

The effect of iron and zinc dietary restriction of pregnant rats on physical growth of litters.

Evidence suggests that micronutrient deficiencies may be associated with problems in early growth. Iron (Fe) and Zinc (Zn) deficiency (D) are prevalent during gestation in low-income countries. For pregnant dams, adequate amount of these micronutrients are needed in the diet to ensure the capacity for increased physical growth. In this study, the role of Fe and Zn dietary restriction of pregnant rats on physical growth of litters was investigated. Pregnant rats after to mating were divided to three groups. Control group fed a standard diet and a FeD group fed a diet deficient in Fe and a ZnD group fed a diet deficient in Zn. All the diets were exposed during the last third of pregnancy. The results showed serum Fe and Zn concentration after to exert dietary compared to before to exert dietary in FeD and ZnD groups was significant. There was a significant difference in the physical growth indexes (body weight, body length, tail length, and head length) between FeD and ZnD groups compared to the Control group, but a significant difference in head width and brain weight between FeD and ZnD groups compared to the Control group was not seen. The results of this study suggest that adequate Fe and Zn affect the physical growth of litters.

Effect of reversible inactivation of locus ceruleus on spatial reference and working memory.

The locus coeruleus (LC) is the largest source of norepinephrine (NE) in the prefrontal cortex and the hippocampus, influencing the cognitive functions of these areas. All previous studies have studied the role of the LC-NE system on learning and memory using the irreversible lesion technique, employing either electrocoagulation or excitotoxins. However, the reversible functional inactivation of LC by means of stereotaxic local microinjection of lidocaine could measure the phases of memory processing (acquisition, consolidation and retention) without any interference with the other cognitive functions of the same structure either during earlier or later phases of the same process. The aim of this study is to investigate LC involvement in spatial reference and working memory by inducing bilateral pre-training, post-training and pre-retrieval lidocaine functional inactivation using the Morris water maze task. The reversible inactivation of LC was applied at different stages of spatial memory formation: (1) immediately before the training sessions to determine the effects on acquisition of the both reference and working memory; (2) immediately after the training session to evaluate effects on both spatial memory consolidation and retention of working memory; and (3) immediately before the 24 h retention session to analyze the effects on the retrieval process of reference memory. Our results indicate that the bilateral reversible inactivation of LC significantly impaired the acquisition of reference and working memory, while it had no effect on consolidation and/or retention of such memories in the Morris water maze (MWM) task. Therefore, the noradrenergic system of the LC may play a more important role in acquisition than in consolidation and retrieval of spatial memory in wistar rats.

The effect of spatial learning on the number of astrocytes in the CA3 subfield of the rat hippocampus.

INTRODUCTION: Astrocytes play an important role in the hippocampus, probably in memory and learning. The aim of this study was to evaluate the number of astrocytes in the CA3 subfield of the rat hippocampus after spatial learning using the Morris water maze with reference and working memory methods. METHODS: 45 male albino wistar rats were divided into three groups, with 15 rats in the control group and 15 rats in each of the other two groups. The two study groups of rats underwent spatial learning using the Morris water maze, with one group trained using the reference memory and the other, the working memory technique, respectively. After histological processing, the slides of the brains were stained with the phosphotanguestic acid haematoxylin staining method for detection of the astrocytes. RESULTS: There was a significant difference in the number of astrocytes in the CA3 area between the control and reference memory groups. The difference between control and working memory groups was significant as well. Additionally, when comparing the two learning groups, we also found significant differences between them. CONCLUSION: The number of astrocytes increased due to spatial learning.

Involvement of nitric oxide in insulin induced memory improvement.

Although brain was considered as an insulin-insensitive organ, recently it has appeared that insulin has some interesting effects on some brain regions like hippocampus. It has been known that intra-hippocampally administered insulin can improve learning and memory. Knowing that insulin can stimulate nitric oxide (NO) synthesis via eNOS activation and also that NO synthase (NOS) inhibitors can affect learning and memory, the aim of this study was to assess if NO is involved in insulin induced memory improvement. Wistar male rats were intra-CA1 cannulated and the effect of post-training and pre-probe trial intra-hippocampal administration of N-nitro-L-arginine methyl ester (L-NAME) (5, 10, 30 microg), insulin+L-NAME+/-L-arginine were assessed in a single-day testing version of Morris water maze (MWM) task. Our results show that, l-NAME can prevent insulin induced memory improvement. This drug had no effect on escape latency of a non-spatial visual discrimination task. Therefore, it seems that endogenous nitric oxide has a role in spatial learning and memory improvement caused by insulin.

Intra CA1 insulin microinjection improves memory consolidation and retrieval.

Although the brain was considered as an insulin-insensitive organ, recent studies have shown that insulin receptors exist in the brain and insulin modulates some of the brain tasks. Insulin and its receptor are found in specific areas of CNS with a variety of region-specific functions different from its direct glucose regulation in the periphery. The hippocampus and cerebral cortex distributed insulin/insulin receptor has been shown to be involved in brain cognitive functions. The improving effect of insulin on spatial memory acquisition has been shown. In the present study, the effect of insulin microinjection into the CA1 region of rat hippocampus on spatial memory consolidation and retrieval has been investigated. Insulin in 12 MU (but not in 0.5 and 6 MU) improved both memory retrieval and consolidation.

Insulin protects against stress-induced impairments in water maze performance.

The presence of insulin receptor in the hippocampus suggests that this organ is a target for insulin. However, unlike the classic peripheral insulin target tissues such as adipocyte, muscle and liver, where the primary function of insulin is to regulate glucose homeostasis, insulin in the central nervous system (CNS) exhibits more diverse actions, most of which have not been clearly understood. A direct role of hippocampal insulin receptor signaling in improving cognitive functions, including learning and memory, and the association of insulin receptor deterioration with brain degenerative dementia (e.g., Alzheimer's disease) have attracted increasing interest. Additionally it has been shown that insulin can be a neuroprotective agent against memory loss induced by ischemia, lesions and some pharmacological agents. In the present study we evaluate the hypothesis that the bilateral intra CA1 insulin injection can protects against stress-induced memory deficit. Chronic restraint stress (2h per day x 7 days) significantly impaired spatial performance in Morris water maze and elevated serum corticosterone level. Intrahippocampal insulin microinjection was done 15-20 min before every stress episode. Insulin in low dose (0.5 MU) had no significant effect on memory deficit induced by stress. But in higher doses (6 and 12 MU) insulin protects animals against the deleterious effect of stress. Insulin alone daily injection had no effect on water maze performance. These results suggest that spatial learning and memory is compromised during chronic stress and insulin may protect against this effect.

The astrocytes number in different subfield of rat's hippocampus in reference memory learning method.

In this study with usage of morris water maze and reference memory technique, we used 10 male albino wistar rats. Five rats in control group and 5 rats in Reference memory group. After histological preparation, the slides were stained with PTAH staining for showing the Astrocytes. Present results showed significant difference in astrocytes number in CA1, CA2 and CA3 area of hippocampus between control and reference memory group. The number of astrocytes is increased in reference memory group. Then we divided the hippocampus to three parts: Anterior, middle and posterior and with compare of different area (CA1, CA2 and CA3) of hippocampus, we found that the increase of astrocytes number in posterior two-third of CA2 and CA3 is more than of it's number in the anterior one-third.

The similarity of astrocytes number in dentate gyrus and CA3 subfield of rats hippocampus.

The dentate gyrus is a part of hippocampal formation that it contains granule cells, which project to the pyramidal cells and interneurons of the CA3 subfield of the hippocampus. Astrocytes play a more active role in neuronal activity, including regulating ion flux currents, energy production, neurotransmitter release and synaptogenesis. Astrocytes are the only cells in the brain that contain the energy molecule glycogen. The close relationship between dentate gyrus and CA3 area can cause the similarity of the number of astrocytes in these areas. In this study 5 male albino wistar rats were used. Rats were housed in large plastic cage in animal house and were maintained under standard conditions, after histological processing, The 7 microm slides of the brains were stained with PTAH staining for showing the astrocytes. This staining is specialized for astrocytes. We showed that the number of astrocytes in different (ant., mid., post) parts of dentate gyrus and CA3 of hippocampus is the same. For example, the anterior parts of two area have the most number of astrocytes and the middle parts of two area have the least number of astrocytes. We concluded that dentate gyrus and CA3 area of hippocampus have the same group of astrocytes.

The effect of intrahippocampal insulin microinjection on spatial learning and memory.

Insulin is best known for its action on peripheral target tissues such as the adipocyte, muscle and liver to regulate glucose homeostasis. Insulin and its receptor are found in specific area of CNS with a variety of region-specific functions different from its direct glucose regulation in the periphery. The hippocampus and cerebral cortex distributed insulin/insulin receptor has been shown to be involved in brain cognitive functions. Previous studies about the effect of insulin on memory are controversial. In the present study, the effect of insulin microinjection into CA1 region of rat hippocampus on water maze performance has been investigated. Insulin had a discrepant effect dose dependently. The spatial learning and memory were impaired with lower dose of insulin, had not changed with intermediate doses, while they improved with higher doses. These results suggest that insulin may have a dose-dependent effect on spatial learning and memory.

Microinjection of ritanserin into the dorsal hippocampal CA1 and dentate gyrus decrease nociceptive behavior in adult male rat.

Prenatal 5HT depletion causes a significant decrease in the level of nociceptive sensitivity during the second phase of the formalin test behavioral response. These experiments were designed to test whether blocking 5HT2A/2c receptors in the CA1 region of the hippocampus and dentate gyrus would decrease nociceptive behaviors induced by a peripheral noxious stimulus formalin as an animal model of unremitting human being. The 5HT2A/2c receptor antagonist ritanserin (2, 4 and 8 microg/0.5 microl) was injected into the CA1 area and dentate gyrus of behaving rats 5 min before subcutaneous injection of formalin irritant. Nociceptive behaviors in both phases of the formalin test were significantly decreased by ritanserin (4 and 8 microg/0.5 microl) and ritanserin had no effect at 2 microg/0.5 microl. These results support the hypothesis that the hippocampal formation may modify the processing of incoming nociceptive information and that 5HT2A/2c receptor-sensitive mechanisms in the hippocampus may play a role in nociception and/or the expression of related behaviors.

Impaired spatial learning in the Morris water maze induced by serotonin reuptake inhibitors in rats.

The effects of selective serotonin reuptake inhibitors citalopram and fluoxetine on spatial learning were assessed in rats. Adult male rats were subjected to 4 days of training in the Morris water maze with the invisible platform. Animals received different doses of citalopram (1-8 mg/kg; i.p.) or fluoxetine (1-16 mg/kg; i.p.) or their vehicles (saline or distilled water respectively) 30 minutes before training each day. The results showed that citalopram at doses of 4 and 8 mg/kg and fluoxetine at doses of 8 and 16 mg/kg significantly increased latencies to find the platform and traveled distances compared to the control group. Therefore, it appears that selective serotonin reuptake inhibitors can cause learning deficits in complex spatial tasks such as Morris water maze.

The effects of intrahippocampal testosterone and flutamide on spatial localization in the Morris water maze.

The high density of the androgen receptors in fundamental centers of learning and memory, such as hippocampus, shows that there must be some relationships between the androgen receptors and cognitive aspects. To determine the role of hippocampal androgen receptors in spatial learning, the current research has been conducted to assess the effect of testosterone enanthate, as the agonist, and flutamide, as the antagonist, of these receptors on spatial discrimination of rats, using the Morris water maze (MWM). Adult male rats were bilaterally cannulated into the CA1 region of their hippocampus. Different groups received different doses of flutamide (2, 5, 10 and 20 microg/0.5 microl) or testosterone enanthate (20, 40 and 80 microg/0.5 microl) through the cannulas 30 min before training for 3 days. The results showed dose-dependent increases in latencies and traveled distances to find the invisible platform both in flutamide- and testosterone-treated groups as compared to the control group, with peak effects at doses of 5 microg/0.5 microl for flutamide and 80 microg/0.5 microl for testosterone. Therefore, it seems that both androgen receptor blockade and exogenous testosterone can effect spatial localization of adult, male rats.

Lidocaine reversible inactivation of the median raphe nucleus has no effect on reference memory but enhances working memory versions of the Morris water maze task.

Numerous studies in the past have dealt with the role of serotonergic system lesions in tasks aimed at measurement of cognitive behavior, but the literature concerning the role of serotonin in cognition remains controversial. Rats with electrolytic lesions of the median raphe nucleus (MRN) were found to display a profound impairment in both the acquisition and retention of spatial memory task. In this study, the lidocaine inactivation was employed to evaluate the involvement of the rat's median raphe nucleus in reference and working memory versions of the Morris water maze (MWM) task. Lidocaine (0.5 microl, 2%) was injected through a single cannula aimed at the MRN; control groups were treated in the same way with a 0.5 microl injection of saline. In Experiment 1, rats were trained in a reference memory version of the MWM with two blocks of four trials per day for three consecutive days, with intra-cerebral injection made 5 min before training. No significant difference was found. In Experiment 2, intra-cerebral injection was applied immediately after two blocks of four trials, and in Experiment 3, the drug was injected 5 min before retention test in rats that had received eight trials per day on three consecutive days. Again, no significant difference between control and treatment groups was found. These results indicate that MRN has no role in acquisition, consolidation and retrieval of spatial reference memory. In subsequent experiments, rats were trained in a working memory version of the MWM task to find a new target position in trial 1, and retrieval was tested 75 min later. MRN inactivation 5 min before (Experiment 4) and immediately after the acquisition trial (Experiment 5) enhanced spatial working memory. It is concluded that normal activity of the MRN has no role in formation and retrieval of reference memory, but it has an inhibitory role in working memory. Our results are confirmed with other studies suggesting that the serotonergic system has a different role in long-term and short-term memory. Interaction with other neurotransmitter systems like acetylcholine may be involved in this case.

Reversible inactivation of the median raphe nucleus enhances consolidation and retrieval but not acquisition of passive avoidance learning in rats.

Involvement of median raphe nucleus (MRN) in acquisition, consolidation and retrieval of passive avoidance (PA) was investigated with functional suppression of this area by lidocaine. Rats carrying a chronically implanted cannula aimed at the MRN were trained on a step-through passive avoidance task and received intra-MRN injection of lidocaine or saline 5 min before training or 5, 90 and 360 min after acquisition trial or 5 min before the retrieval test. Lidocaine MRN inactivation had no effect on PA learning. Lidocaine injected 5 and 90 min after the acquisition trial significantly enhanced avoidance of the dark compartment in comparison with the control group injected with saline. But PA retention was not affected by lidocaine injected 360 min after acquisition or 5 min before training. Retention latency significantly increased, when lidocaine injected 5 min before retrieval test. Step-through latency of naive rats was not affected by MRN blockade. Furthermore, reversible inactivation of MRN did not have a significant effect on locomotor activity. Our results indicate that the MRN contributes to PA consolidation at least until 90 min after acquisition and involves in PA retrieval. It is concluded that functional ablation of the MRN may disrupt the inhibitory actions of MRN projections to sub-cortical circuits participating in PA memorization and retrieval.