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Insulin protects against stress-induced impairments in water maze performance
Behavioural Brain Research 176 (2007) 230–236
Research report
Insulin protects against stress-induced impairments in water maze performance M. Moosavi a,c , N. Naghdi b , N. Maghsoudi c , S. Zahedi Asl d,∗ a
Department of Physiology, School of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran b Department of Physiology, Pasteur Institute of Iran, Tehran, Iran c Neuroscience Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran d Endocrine Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran Received 17 August 2006; received in revised form 28 September 2006; accepted 6 October 2006 Available online 20 November 2006
Abstract 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 (2 h per day × 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Insulin; Hippocampus; Neuroprotection; Memory; Morris water maze; Stress; Corticosterone
1. Introduction Chronic stress is known to increase the levels of adrenal glucocorticoids resulting in deleterious cognitive functioning [21,24,32,18]. The hippocampus, a critical structure for spatial learning and memory, is particularly vulnerable to stress-induced glucocorticoid damage [44,22,26,25,32], reflected as deficits in spatial memory tasks [23,38] and synaptic plasticity [37,20]. Prolonged or excessive exposure to glucocorticoids leads to neuronal damages, particularly in the hippocampus, a region enriched with corticosteroid receptors [40]. ∗ Corresponding author at: Endocrine Research Center, Shaheed Beheshti University of Medical Sciences, P.O. Box: 19395-4763, Tehran, IR, Iran. Tel.: +98 21 22409309; fax: +98 21 22402463. E-mail address: [email protected] (S. Zahedi Asl).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.10.011
The mechanism underlying stress-induced neurotoxicity remains unclear, although a number of studies suggest that glutamate excitotoxicity plays a primary role [35]. Another aspect of the negative action of glucocorticoids on neuronal homeostasis is the disruption of neuronal protective mechanisms such as feedback regulation of Ca2+ channel functions [10]. It thus seems that glucocorticoids can not only induce direct deleterious effects but also can impair neuroprotective components of neuronal survival. Insulin and its receptor are dispersed throughout the brain with the highest density located in the cerebral cortex and hippocampus [14,45,3,43]. The hippocampus expresses insulin messenger RNA and has a high number of insulin receptors [36,47,48]. It has been showed that insulin receptor is selectively enriched in CA1 region of hippocampus [5]. Insulin appears to have some neuroprotective effects against memory loss induced by ischemia, lesions and pharmaco-
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logical inhibition [8,4,33]. Insulin can activate several signaling pathways [28,42,3], among which the insulin receptor substrate-1 (IRS-1)/PI-3 kinase/phosphoinositide-dependent kinase (PDK)/protein kinase B (PKB/Akt) has been most intensively studied [3]. Akt/PKB serves a key role in mediating anti-apoptotic actions of growth factors on cell [7] and plays an important role in neuronal protection. Considering the neuroprotective action of insulin and the deleterious effects of stress particularly on hippocampal neurons, in the present study we examined the hypothesis that spatial learning and memory deficits, induced by chronic immobilization stress can be attenuated through intrahippocampal microinjection of insulin. 2. Materials and methods 2.1. Animals and substances Adult male albino Wistar rats (300–350 g, 5–6 months age randomly assigned into the groups) obtained from Pasteur Institute of Iran were housed in a temperature (25 ± 2 ◦ C) and humidity-controlled room. The animals were maintained under a 12:12 h light/dark cycle, with lights off at 7:00 p.m. Food and water provided ad libitum except for the periods of behavioral testing in Morris water maze (MWM) and stress induction. All experimental procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The animals were randomly assigned into the groups (10 in each) and all tests were carried out during the light phase. Groups were including stress and insulin treatment (0.5, 6 and 12 MU) (stress-insulin), stress treatment and saline microinjection (stress-saline), insulin (0.5, 6 and 12 MU) microinjection without stress treatment (control-insulin) and saline injection without any stress treatment (control-saline). Insulin was purchased from Exir Pharmaceutical Company (Borujerd, Iran) and was diluted in sterile 0.9% saline. Insulin dose was selected according to our previous study in that we suggested insulin can have a dose dependent effect on learning ability. In the low dose (0.5 MU) insulin had a slight impairing effect, with intermediate doses it had not any significant effect, while in the higher doses it had a strong improving effect on spatial learning and memory [27]. In the present study we chose three doses each one from those three categories (low, intermediate and the higher doses).
2.2. Surgery The rats were implanted with bilateral canula aimed at the dorsal hippocampus. Before surgery animals were anesthetized with IP injections of ketamine (100 mg/kg body weight, Gedoon Richter, Budapest, Hungary) and xylazine (10 mg/kg body weight, Bayer AG, Leverkusen, Germany). The animals were mounted into a stereotaxic frame used to position the 22-gauge stainless steel guide canula in the dorsal hippocampus (AP −3.8, L ±2.2, V −2.7). Coordinates were chosen based on a rat brain atlas [30]. The internal canula was 0.5 mm longer than guide canula. The canula was anchored to the skull using stainless steel screws and acrylic cement.
2.3. Immobilization stress and corticosterone assay To induce the stress, animals were immobilized in a plastic rodent restrainer and secured in the dorsal recumbent position for 2 h/day for 7 days (days 5–11 after surgery), at 11:00–13:00 h in a neutral cage [32]. After light ether anesthesia, blood samples were taken by tail snipping on the first and seventh days of the stress treatment at 11:00 h (baseline) and 13:00 h (end of the stress session). One millilitre of blood samples was collected in an Eppendorf tube and centrifuged at 5000 rpm for 10 min at 4 ◦ C. Serum was removed and kept at −70 ◦ C to quantify corticosterone levels using a commercial rat corticosterone radioimmunoassay kit (DRG, Germany). Intra- and inter-assay coefficients of variations for corticosterone measurements were less than 10%.
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2.4. Apparatus The water maze was a black circular pool with a diameter of 136 cm and a height of 60 cm, filled with 20 ± 1 ◦ C water to a depth of 20 cm. The maze was divided geographically into four equal quadrants and release points that were designed at each quadrant as N, E, S, and W. A hidden circular platform (10 cm in diameter), made of Plexiglas, was located in the center of the southwest quadrant, submerged 1.5 cm beneath the surface of the water. Fixed, extra maze visual cues were present at various locations around the maze (i.e. computer, MWM hard wares, posters). An infrared camera was mounted above the center of the maze. An infrared LED was attached to each rat as a probe so that the animal motion can be recorded and sent to the computer. A tracking system was used to measure the escape latency, traveled path and swimming speed [27].
2.5. Microinjection procedure The microinjections were made using a 10 l Hamilton syringe through a short piece of polyethylene tube. The needle was inserted 0.5 mm beyond the tip of the canula. Saline or insulin (0.5, 6 and 12 MU) were injected (0.5 l) into bilateral CA1 region during 2 min by a microinjection pump and the needle was left in the place for 1 min following the microinjections to minimize the flow back of the solution. Fifteen minutes after the intrahippocampal injection of insulin or saline stress treatment was started.
2.6. Behavioral procedure On day 12 after surgery (1 day after stress treatment completion), the rats were trained in the water maze. The single training session consisted of eight trials with four different starting positions that were equally distributed around the perimeter of the maze [9,29,6,1,27]. The task requires rats to swim to the hidden platform guided by distal spatial cues. After mounting the platform, the rats were allowed to remain there for 20 s, and were then placed in a holding cage for 30 s until the start of the next trial. Rats were given a maximum of 60 s to find the platform and if it failed to find the platform in 60 s, it was placed on the platform and allowed to rest for 20 s. Latency to platform and distance traveled were collected and analyzed later. After completion of the training, the animals were returned to their home cages until retention testing 24 h later. The probe trial consisted of a 60 s free swim period without a platform and the time swum in the target quadrant was recorded.
2.7. Histology At the completion of behavioral training and blood sampling, animals were killed and their brain were removed and stored in 10% formalin. Two days later the brain sectioned and canula placements were examined for verification of needle tip locations (Fig. 1).
2.8. Experimental groups 2.8.1. Experiment 1 The aim of this experiment was to evaluate the effect of stress on serum corticosterone level. In addition it was evaluated whether the intrahippocampal injection of insulin in three doses (0.5, 6 and 12 MU) can have any effect on serum corticosterone level or not. The intrahippocampally canulated rats were randomly divided into 4 groups (10 rats in each): stressed-saline group (group B), stressed-0.5 insulin (group C), stressed-6 insulin group (group D) and stressed12 insulin (group E). Saline or insulin was injected intrahippocampally (bilateral) 15–20 min before each stress episode. After light ether anesthesia blood samples were taken by tail snipping on the first and seventh days of the stress treatment at 11:00 h (before stress as baseline) and 13:00 h (end of the stress session) in each group. 2.8.2. Experiment 2 The aim of this experiment was to evaluate the effect of stress on spatial learning and memory. The canulated stressed-saline and control-saline animals were trained 1 day after stress and/or saline treatment completion in the Morris water maze for eight trials. The escape latency and distance traveled were
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3. Results 3.1. Experiment 1 Serum corticosterone levels were measured in the rats at baseline (11:00 h) and immediately following the immobilization treatment (13:00 h) on the first and last days of the stress treatment. As was expected, stressed rats had significantly elevated levels of corticosterone at the post-stress time (Table 1, F15,96 = 21.915 P < 0.0001). Bilateral intrahippocampal injection of insulin with all three doses (0.5, 6 and 24 MU) did not have any effect on corticosterone level before or/and after stress. Additionally no significant difference was found within groups for any day tested at either baseline or post-stress time points. 3.2. Experiment 2 Fig. 1. Nissl-stained coronal brain section from canulated and injected rats. The canula and the injection position are shown. recorded to analyze later. The retention testing was done 24 h later as a 60 S probe trial. 2.8.3. Experiment 3 The aim of this experiment was to evaluate the neuroprotective effect of insulin against stress-induced spatial learning and memory deficit. The canulated stressed-insulin (0.5, 6 and 12 MU) animals were trained 1 day after stress treatment completion in the Morris water maze for eight trials. The escape latency and distance traveled were recorded to analyze later. The retention testing was done 24 h later as a 60 S probe trial. 2.8.4. Experiment 4 The aim of this experiment was to evaluate the effect of daily injection of saline or insulin without any stress on water maze performance. The intrahippocampally canulated animals were divided randomly into four groups (10 in each): control-saline (group A), control-insulin 0.5 (group F), control-insulin 6 (group G) and control-insulin 12 (group H). Saline or insulin was injected intrahippocampally for 7 days (daily injection). The animals were trained 1 day after saline or insulin treatment completion in the Morris water maze for eight trials. The escape latency and distance traveled were recorded to analyze later. The retention testing was done 24 h later as a 60 S probe trial and the time swum in the target quadrant was recorded.
2.9. Statistical analysis Data are expressed, as means ± S.E.M. The statistical analysis of the data was carried out by one-way ANOVA-followed by Student’s Newman Keuls test- or unpaired t-test as required. In all comparisons, P < 0.05 was considered significant.
Chronic immobilization stress (stress-saline) significantly (t18 = 3.575, P = 0.0022) increased escape latency comparing with saline-treated unstressed animals (Fig. 2A and B). Stress significantly (t18 = 3.360, P = 0.0035) increased distance traveled comparing with saline-control animals (Fig. 2C). The results of probe test have been shown in Fig. 2D. In the probe trial, stress-saline animals spent less time in the target quadrant than did control-saline animals, indicating memory deficits in these animals (t18 = 3.493, P = 0.0026). There was no significant difference in the swimming speed between these groups (data not shown). 3.3. Experiment 3 The neuroprotective effect of insulin against stress-induced spatial learning and memory deficit has been assessed. Insulin with low dose (0.5 MU insulin + stress) had no significant effect on escape latency comparing with stress-saline group. Animals treated with higher doses of insulin (6 and 12 MU insulin + stress) demonstrated better spatial learning than that of the saline treated animals (stress-saline) as latency to platform was significantly (F3,36 = 9.430, P < 0.0001) shorter in this groups(Fig. 3A and B). One-way ANOVA of the distance traveled revealed significant differences between these groups (F3,36 = 8.088, P = 0.0003). Low dose insulin (0.5 MU) had no significant effect on the distance traveled comparing with stresssaline group, but 6 and 12 MU of insulin treatment in stressed
Table 1 The effect of stress on serum corticosterone levels at baseline (11:00 h) and immediately following the immobilization treatment (13:00 h) on the first and last days of the stress treatment Groups
Corticosterone (ng/ml) First day
Seventh day
Before stress Stress-saline Stress-0.5 MU insulin Stress-6 MU insulin Stress-12 MU insulin ** P < 0.01
113 138.9 130 124.6
± ± ± ±
19 29.7 30.1 17.5
After stress 569.5 598.1 580.2 507.8
± ± ± ±
50*** 67.3*** 50.1*** 24***
Before stress 323.3 364.5 301.2 326
and *** P < 0.001 indicate the difference vs. first day in each group. Values are mean ± S.E.M. of seven rats.
± ± ± ±
22.5** 44.2*** 34.1** 22.4**
After stress 476 538.1 476 456
± ± ± ±
42*** 73.9*** 37.1*** 20***
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Fig. 2. The effect of immobilization stress on water maze performance. (A) The learning curves. (B) Average escape latency. (C) Average distance traveled. (D) The probe trial. ** P < 0.01 indicates the difference between stress-saline (B) and control-saline (A) group.
animals decreased the distance traveled significantly (Fig. 3C). One-way ANOVA of the probe trial revealed significant differences between these groups (F3,36 = 20.171, P < 0.0001). Animals treated with 0.5 MU of insulin (0.5 MU insulin + stress) show no significant difference comparing with saline-stress group. But stressed animals treated with 6 and 12 MU of insulin significantly spent more time in the target quadrant than did saline-stress group, indicating memory improvement in these animals (Fig. 3D). There was no significant difference in the swimming speed between these groups (data not shown). 3.4. Experiment 4 The animals treated with insulin without any stress (controlinsulin), had not showed any significant effect in comparison with saline-control group in escape latency, distance traveled or probe trial (Fig. 4). In addition there was no significant difference in the swimming speed between these groups and control-saline group (data not shown). 4. Discussion This study examined the putative role of insulin in stressinduced spatial learning and memory deficits. Chronic immobi-
lization stress paradigm significantly elevated circulating corticosterone levels and resulted in impairment of spatial learning and memory on the Morris water maze. The stressed animals treated with insulin displayed no such dysfunction and actually performed similar to non-stressed controls. This is in spite of the fact that corticosterone levels were similarly elevated in all groups during the stress paradigm. These results demonstrate that spatial learning and memory, which are compromised during chronic stress, may be dependent on insulin for normal functioning. The similar speed of animals indicates that stress treatment and (or) intrahippocampal insulin injection had no effect on locomotor’s behavior. It has been shown that chronic immobilization stress leads to increased circulating levels of corticosterone resulting in impairment of spatial learning and memory on the Morris water maze and long term potentiation in the CA1 region of hippocampal slices taken from stressed rats [32]. The intracellular mechanisms involved in corticosterone-induced memory deficit, however, remain to be fully established. It has been suggested that corticosterone could disrupt protective mechanisms such as the feedback regulation of Ca2+ channel functions. Increased Ca2+ levels induced by corticosterone may promote excessive glutamate release, leading to excitotoxicity [12,19]. Glutamate and
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Fig. 3. The effect of intra-CA1 insulin microinjection on stress-induced memory deficit. (A) The learning curves. (B) Average escape latency. (C) Average distance traveled. (D) The probe trial. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicate the difference between stress-saline and stress-insulin group. Stress-saline (B), stress-0.5 insulin (C), stress-6 insulin (D), stress-12 insulin (E).
intracellular influx of Ca2+ have been shown to induce neuronal death via the inhibition of Akt kinase [31]. In this study insulin was protective against stress-induced deleterious effect on learning and memory. Despite the increase in corticosterone levels during immobilization stress, insulin-treated animals displayed normal learning and memory. Indices of spatial learning showed these animals acquired and retained the information better than saline-injected animals. Several signaling pathways activated by insulin receptor have been identified [28,42,3], among which the insulin receptor substrate-1 (IRS-1)/PI-3 kinase/phosphoinositide-dependent kinase (PDK)/protein kinase B (PKB/Akt) has been most intensively studied. Akt/PKB, a 57-kDa protein–serine/threonine kinase [11], serves a key role in mediating anti-apoptotic actions of growth factors on cell [7] and plays an important role in neuronal protection. Insulin may protect hippocampal cells against stress-induced deleterious effects through this mechanism. Alternatively, the immobilization paradigm used in this study could possibly induce a state of learned helplessness or depression in the animals. It may be that our stressor simply caused a state of depression in the animals, which was ameliorated by insulin treatment, since it has been showed that intranasal administration of insulin to human subjects can improve their mood [2]. However the exact mechanism of this insulin protective effect is remained to be elucidated.
The intrahippocampal injection of insulin had not any effect on serum corticosterone level as seen it Table 1 (the corticosterone level after insulin injection and before stress session at first and seventh days of treatment). The animals treated with insulin without any stress (insulincontrol), had not showed any significant effect in comparison with saline-control group. It showed that the protective effect of insulin is independent of its direct effect on learning and memory, which had been seen with acute insulin injection [27]. Consistent with our finding, insulin treatment, immediately following ischemia, can prevent ischemia-induced learning deficits. The insulin treatment also significantly reduced the CA1 neuronal necrosis caused by ischemia [39,33]. Also insulin pretreatment before dorsal hippocampal lesions induction, significantly reduced memory deficits for active avoidance learning experience in the rat [8]. In pharmacological experiments, insulin was shown to overcome scopolamine-induced memory impairment in rats trained in a radial arm maze task [4]. On the other hand the deterioration of CNS insulin receptor functions is associated with the pathogenesis of aging-related brain degenerative dementias such as sporadic Alzheimer’s disease [15–17,41,34]. Brain insulin and insulin receptor are reduced in the brains of sporadic Alzheimer’s disease patients [13]. Consistent results have also been found in rodents whose brains show aging-reduced insulin receptor numbers [46].
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Fig. 4. The effect of intra-CA1 daily microinjection of insulin on water maze performance. (A) The learning curves. (B) Average escape latency. (C) Average distance traveled. (D) The probe trial. Control-saline (A), control-0.5 insulin (F), control-6 insulin (G), control-12 insulin (H).
In our previous study we demonstrated that pre-training microinjection of insulin to CA1 region of the hippocampus can affect learning and memory in a dose-dependent manner. With lower dose (0.5 MU) insulin slightly impaired learning, with intermediate doses it had no significant effect on it and with higher doses it significantly improved spatial learning and memory. However the impairing effect of the lower dose was much weaker than the improving effects of the higher doses [27]. In conclusion, the current study examined the hypothesis that stress-induced deficit in spatial learning and memory can be prevented by intrahippocampal insulin injection. While the results from our behavioral experiments seem to implicate insulin as an influential factor in memory and hippocampal plasticity, much remains to be learned about the specific cellular and molecular mechanisms involved. These results demonstrate the effectiveness of insulin as a key neuro-protective agent. Acknowledgments This work was supported by a grant from Neuroscience Research Center of Shaheed Beheshti University of Medical Sciences. Help from Ms. Shiva for the preparation of the manuscript and Mr. Akbari for technical assistance are very much appreciated.
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Research report
Insulin protects against stress-induced impairments in water maze performance M. Moosavi a,c , N. Naghdi b , N. Maghsoudi c , S. Zahedi Asl d,∗ a
Department of Physiology, School of Medicine, Shaheed Beheshti University of Medical Sciences, Tehran, Iran b Department of Physiology, Pasteur Institute of Iran, Tehran, Iran c Neuroscience Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran d Endocrine Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran Received 17 August 2006; received in revised form 28 September 2006; accepted 6 October 2006 Available online 20 November 2006
Abstract 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 (2 h per day × 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. © 2006 Elsevier B.V. All rights reserved. Keywords: Insulin; Hippocampus; Neuroprotection; Memory; Morris water maze; Stress; Corticosterone
1. Introduction Chronic stress is known to increase the levels of adrenal glucocorticoids resulting in deleterious cognitive functioning [21,24,32,18]. The hippocampus, a critical structure for spatial learning and memory, is particularly vulnerable to stress-induced glucocorticoid damage [44,22,26,25,32], reflected as deficits in spatial memory tasks [23,38] and synaptic plasticity [37,20]. Prolonged or excessive exposure to glucocorticoids leads to neuronal damages, particularly in the hippocampus, a region enriched with corticosteroid receptors [40]. ∗ Corresponding author at: Endocrine Research Center, Shaheed Beheshti University of Medical Sciences, P.O. Box: 19395-4763, Tehran, IR, Iran. Tel.: +98 21 22409309; fax: +98 21 22402463. E-mail address: [email protected] (S. Zahedi Asl).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.10.011
The mechanism underlying stress-induced neurotoxicity remains unclear, although a number of studies suggest that glutamate excitotoxicity plays a primary role [35]. Another aspect of the negative action of glucocorticoids on neuronal homeostasis is the disruption of neuronal protective mechanisms such as feedback regulation of Ca2+ channel functions [10]. It thus seems that glucocorticoids can not only induce direct deleterious effects but also can impair neuroprotective components of neuronal survival. Insulin and its receptor are dispersed throughout the brain with the highest density located in the cerebral cortex and hippocampus [14,45,3,43]. The hippocampus expresses insulin messenger RNA and has a high number of insulin receptors [36,47,48]. It has been showed that insulin receptor is selectively enriched in CA1 region of hippocampus [5]. Insulin appears to have some neuroprotective effects against memory loss induced by ischemia, lesions and pharmaco-
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logical inhibition [8,4,33]. Insulin can activate several signaling pathways [28,42,3], among which the insulin receptor substrate-1 (IRS-1)/PI-3 kinase/phosphoinositide-dependent kinase (PDK)/protein kinase B (PKB/Akt) has been most intensively studied [3]. Akt/PKB serves a key role in mediating anti-apoptotic actions of growth factors on cell [7] and plays an important role in neuronal protection. Considering the neuroprotective action of insulin and the deleterious effects of stress particularly on hippocampal neurons, in the present study we examined the hypothesis that spatial learning and memory deficits, induced by chronic immobilization stress can be attenuated through intrahippocampal microinjection of insulin. 2. Materials and methods 2.1. Animals and substances Adult male albino Wistar rats (300–350 g, 5–6 months age randomly assigned into the groups) obtained from Pasteur Institute of Iran were housed in a temperature (25 ± 2 ◦ C) and humidity-controlled room. The animals were maintained under a 12:12 h light/dark cycle, with lights off at 7:00 p.m. Food and water provided ad libitum except for the periods of behavioral testing in Morris water maze (MWM) and stress induction. All experimental procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The animals were randomly assigned into the groups (10 in each) and all tests were carried out during the light phase. Groups were including stress and insulin treatment (0.5, 6 and 12 MU) (stress-insulin), stress treatment and saline microinjection (stress-saline), insulin (0.5, 6 and 12 MU) microinjection without stress treatment (control-insulin) and saline injection without any stress treatment (control-saline). Insulin was purchased from Exir Pharmaceutical Company (Borujerd, Iran) and was diluted in sterile 0.9% saline. Insulin dose was selected according to our previous study in that we suggested insulin can have a dose dependent effect on learning ability. In the low dose (0.5 MU) insulin had a slight impairing effect, with intermediate doses it had not any significant effect, while in the higher doses it had a strong improving effect on spatial learning and memory [27]. In the present study we chose three doses each one from those three categories (low, intermediate and the higher doses).
2.2. Surgery The rats were implanted with bilateral canula aimed at the dorsal hippocampus. Before surgery animals were anesthetized with IP injections of ketamine (100 mg/kg body weight, Gedoon Richter, Budapest, Hungary) and xylazine (10 mg/kg body weight, Bayer AG, Leverkusen, Germany). The animals were mounted into a stereotaxic frame used to position the 22-gauge stainless steel guide canula in the dorsal hippocampus (AP −3.8, L ±2.2, V −2.7). Coordinates were chosen based on a rat brain atlas [30]. The internal canula was 0.5 mm longer than guide canula. The canula was anchored to the skull using stainless steel screws and acrylic cement.
2.3. Immobilization stress and corticosterone assay To induce the stress, animals were immobilized in a plastic rodent restrainer and secured in the dorsal recumbent position for 2 h/day for 7 days (days 5–11 after surgery), at 11:00–13:00 h in a neutral cage [32]. After light ether anesthesia, blood samples were taken by tail snipping on the first and seventh days of the stress treatment at 11:00 h (baseline) and 13:00 h (end of the stress session). One millilitre of blood samples was collected in an Eppendorf tube and centrifuged at 5000 rpm for 10 min at 4 ◦ C. Serum was removed and kept at −70 ◦ C to quantify corticosterone levels using a commercial rat corticosterone radioimmunoassay kit (DRG, Germany). Intra- and inter-assay coefficients of variations for corticosterone measurements were less than 10%.
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2.4. Apparatus The water maze was a black circular pool with a diameter of 136 cm and a height of 60 cm, filled with 20 ± 1 ◦ C water to a depth of 20 cm. The maze was divided geographically into four equal quadrants and release points that were designed at each quadrant as N, E, S, and W. A hidden circular platform (10 cm in diameter), made of Plexiglas, was located in the center of the southwest quadrant, submerged 1.5 cm beneath the surface of the water. Fixed, extra maze visual cues were present at various locations around the maze (i.e. computer, MWM hard wares, posters). An infrared camera was mounted above the center of the maze. An infrared LED was attached to each rat as a probe so that the animal motion can be recorded and sent to the computer. A tracking system was used to measure the escape latency, traveled path and swimming speed [27].
2.5. Microinjection procedure The microinjections were made using a 10 l Hamilton syringe through a short piece of polyethylene tube. The needle was inserted 0.5 mm beyond the tip of the canula. Saline or insulin (0.5, 6 and 12 MU) were injected (0.5 l) into bilateral CA1 region during 2 min by a microinjection pump and the needle was left in the place for 1 min following the microinjections to minimize the flow back of the solution. Fifteen minutes after the intrahippocampal injection of insulin or saline stress treatment was started.
2.6. Behavioral procedure On day 12 after surgery (1 day after stress treatment completion), the rats were trained in the water maze. The single training session consisted of eight trials with four different starting positions that were equally distributed around the perimeter of the maze [9,29,6,1,27]. The task requires rats to swim to the hidden platform guided by distal spatial cues. After mounting the platform, the rats were allowed to remain there for 20 s, and were then placed in a holding cage for 30 s until the start of the next trial. Rats were given a maximum of 60 s to find the platform and if it failed to find the platform in 60 s, it was placed on the platform and allowed to rest for 20 s. Latency to platform and distance traveled were collected and analyzed later. After completion of the training, the animals were returned to their home cages until retention testing 24 h later. The probe trial consisted of a 60 s free swim period without a platform and the time swum in the target quadrant was recorded.
2.7. Histology At the completion of behavioral training and blood sampling, animals were killed and their brain were removed and stored in 10% formalin. Two days later the brain sectioned and canula placements were examined for verification of needle tip locations (Fig. 1).
2.8. Experimental groups 2.8.1. Experiment 1 The aim of this experiment was to evaluate the effect of stress on serum corticosterone level. In addition it was evaluated whether the intrahippocampal injection of insulin in three doses (0.5, 6 and 12 MU) can have any effect on serum corticosterone level or not. The intrahippocampally canulated rats were randomly divided into 4 groups (10 rats in each): stressed-saline group (group B), stressed-0.5 insulin (group C), stressed-6 insulin group (group D) and stressed12 insulin (group E). Saline or insulin was injected intrahippocampally (bilateral) 15–20 min before each stress episode. After light ether anesthesia blood samples were taken by tail snipping on the first and seventh days of the stress treatment at 11:00 h (before stress as baseline) and 13:00 h (end of the stress session) in each group. 2.8.2. Experiment 2 The aim of this experiment was to evaluate the effect of stress on spatial learning and memory. The canulated stressed-saline and control-saline animals were trained 1 day after stress and/or saline treatment completion in the Morris water maze for eight trials. The escape latency and distance traveled were
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3. Results 3.1. Experiment 1 Serum corticosterone levels were measured in the rats at baseline (11:00 h) and immediately following the immobilization treatment (13:00 h) on the first and last days of the stress treatment. As was expected, stressed rats had significantly elevated levels of corticosterone at the post-stress time (Table 1, F15,96 = 21.915 P < 0.0001). Bilateral intrahippocampal injection of insulin with all three doses (0.5, 6 and 24 MU) did not have any effect on corticosterone level before or/and after stress. Additionally no significant difference was found within groups for any day tested at either baseline or post-stress time points. 3.2. Experiment 2 Fig. 1. Nissl-stained coronal brain section from canulated and injected rats. The canula and the injection position are shown. recorded to analyze later. The retention testing was done 24 h later as a 60 S probe trial. 2.8.3. Experiment 3 The aim of this experiment was to evaluate the neuroprotective effect of insulin against stress-induced spatial learning and memory deficit. The canulated stressed-insulin (0.5, 6 and 12 MU) animals were trained 1 day after stress treatment completion in the Morris water maze for eight trials. The escape latency and distance traveled were recorded to analyze later. The retention testing was done 24 h later as a 60 S probe trial. 2.8.4. Experiment 4 The aim of this experiment was to evaluate the effect of daily injection of saline or insulin without any stress on water maze performance. The intrahippocampally canulated animals were divided randomly into four groups (10 in each): control-saline (group A), control-insulin 0.5 (group F), control-insulin 6 (group G) and control-insulin 12 (group H). Saline or insulin was injected intrahippocampally for 7 days (daily injection). The animals were trained 1 day after saline or insulin treatment completion in the Morris water maze for eight trials. The escape latency and distance traveled were recorded to analyze later. The retention testing was done 24 h later as a 60 S probe trial and the time swum in the target quadrant was recorded.
2.9. Statistical analysis Data are expressed, as means ± S.E.M. The statistical analysis of the data was carried out by one-way ANOVA-followed by Student’s Newman Keuls test- or unpaired t-test as required. In all comparisons, P < 0.05 was considered significant.
Chronic immobilization stress (stress-saline) significantly (t18 = 3.575, P = 0.0022) increased escape latency comparing with saline-treated unstressed animals (Fig. 2A and B). Stress significantly (t18 = 3.360, P = 0.0035) increased distance traveled comparing with saline-control animals (Fig. 2C). The results of probe test have been shown in Fig. 2D. In the probe trial, stress-saline animals spent less time in the target quadrant than did control-saline animals, indicating memory deficits in these animals (t18 = 3.493, P = 0.0026). There was no significant difference in the swimming speed between these groups (data not shown). 3.3. Experiment 3 The neuroprotective effect of insulin against stress-induced spatial learning and memory deficit has been assessed. Insulin with low dose (0.5 MU insulin + stress) had no significant effect on escape latency comparing with stress-saline group. Animals treated with higher doses of insulin (6 and 12 MU insulin + stress) demonstrated better spatial learning than that of the saline treated animals (stress-saline) as latency to platform was significantly (F3,36 = 9.430, P < 0.0001) shorter in this groups(Fig. 3A and B). One-way ANOVA of the distance traveled revealed significant differences between these groups (F3,36 = 8.088, P = 0.0003). Low dose insulin (0.5 MU) had no significant effect on the distance traveled comparing with stresssaline group, but 6 and 12 MU of insulin treatment in stressed
Table 1 The effect of stress on serum corticosterone levels at baseline (11:00 h) and immediately following the immobilization treatment (13:00 h) on the first and last days of the stress treatment Groups
Corticosterone (ng/ml) First day
Seventh day
Before stress Stress-saline Stress-0.5 MU insulin Stress-6 MU insulin Stress-12 MU insulin ** P < 0.01
113 138.9 130 124.6
± ± ± ±
19 29.7 30.1 17.5
After stress 569.5 598.1 580.2 507.8
± ± ± ±
50*** 67.3*** 50.1*** 24***
Before stress 323.3 364.5 301.2 326
and *** P < 0.001 indicate the difference vs. first day in each group. Values are mean ± S.E.M. of seven rats.
± ± ± ±
22.5** 44.2*** 34.1** 22.4**
After stress 476 538.1 476 456
± ± ± ±
42*** 73.9*** 37.1*** 20***
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Fig. 2. The effect of immobilization stress on water maze performance. (A) The learning curves. (B) Average escape latency. (C) Average distance traveled. (D) The probe trial. ** P < 0.01 indicates the difference between stress-saline (B) and control-saline (A) group.
animals decreased the distance traveled significantly (Fig. 3C). One-way ANOVA of the probe trial revealed significant differences between these groups (F3,36 = 20.171, P < 0.0001). Animals treated with 0.5 MU of insulin (0.5 MU insulin + stress) show no significant difference comparing with saline-stress group. But stressed animals treated with 6 and 12 MU of insulin significantly spent more time in the target quadrant than did saline-stress group, indicating memory improvement in these animals (Fig. 3D). There was no significant difference in the swimming speed between these groups (data not shown). 3.4. Experiment 4 The animals treated with insulin without any stress (controlinsulin), had not showed any significant effect in comparison with saline-control group in escape latency, distance traveled or probe trial (Fig. 4). In addition there was no significant difference in the swimming speed between these groups and control-saline group (data not shown). 4. Discussion This study examined the putative role of insulin in stressinduced spatial learning and memory deficits. Chronic immobi-
lization stress paradigm significantly elevated circulating corticosterone levels and resulted in impairment of spatial learning and memory on the Morris water maze. The stressed animals treated with insulin displayed no such dysfunction and actually performed similar to non-stressed controls. This is in spite of the fact that corticosterone levels were similarly elevated in all groups during the stress paradigm. These results demonstrate that spatial learning and memory, which are compromised during chronic stress, may be dependent on insulin for normal functioning. The similar speed of animals indicates that stress treatment and (or) intrahippocampal insulin injection had no effect on locomotor’s behavior. It has been shown that chronic immobilization stress leads to increased circulating levels of corticosterone resulting in impairment of spatial learning and memory on the Morris water maze and long term potentiation in the CA1 region of hippocampal slices taken from stressed rats [32]. The intracellular mechanisms involved in corticosterone-induced memory deficit, however, remain to be fully established. It has been suggested that corticosterone could disrupt protective mechanisms such as the feedback regulation of Ca2+ channel functions. Increased Ca2+ levels induced by corticosterone may promote excessive glutamate release, leading to excitotoxicity [12,19]. Glutamate and
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Fig. 3. The effect of intra-CA1 insulin microinjection on stress-induced memory deficit. (A) The learning curves. (B) Average escape latency. (C) Average distance traveled. (D) The probe trial. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicate the difference between stress-saline and stress-insulin group. Stress-saline (B), stress-0.5 insulin (C), stress-6 insulin (D), stress-12 insulin (E).
intracellular influx of Ca2+ have been shown to induce neuronal death via the inhibition of Akt kinase [31]. In this study insulin was protective against stress-induced deleterious effect on learning and memory. Despite the increase in corticosterone levels during immobilization stress, insulin-treated animals displayed normal learning and memory. Indices of spatial learning showed these animals acquired and retained the information better than saline-injected animals. Several signaling pathways activated by insulin receptor have been identified [28,42,3], among which the insulin receptor substrate-1 (IRS-1)/PI-3 kinase/phosphoinositide-dependent kinase (PDK)/protein kinase B (PKB/Akt) has been most intensively studied. Akt/PKB, a 57-kDa protein–serine/threonine kinase [11], serves a key role in mediating anti-apoptotic actions of growth factors on cell [7] and plays an important role in neuronal protection. Insulin may protect hippocampal cells against stress-induced deleterious effects through this mechanism. Alternatively, the immobilization paradigm used in this study could possibly induce a state of learned helplessness or depression in the animals. It may be that our stressor simply caused a state of depression in the animals, which was ameliorated by insulin treatment, since it has been showed that intranasal administration of insulin to human subjects can improve their mood [2]. However the exact mechanism of this insulin protective effect is remained to be elucidated.
The intrahippocampal injection of insulin had not any effect on serum corticosterone level as seen it Table 1 (the corticosterone level after insulin injection and before stress session at first and seventh days of treatment). The animals treated with insulin without any stress (insulincontrol), had not showed any significant effect in comparison with saline-control group. It showed that the protective effect of insulin is independent of its direct effect on learning and memory, which had been seen with acute insulin injection [27]. Consistent with our finding, insulin treatment, immediately following ischemia, can prevent ischemia-induced learning deficits. The insulin treatment also significantly reduced the CA1 neuronal necrosis caused by ischemia [39,33]. Also insulin pretreatment before dorsal hippocampal lesions induction, significantly reduced memory deficits for active avoidance learning experience in the rat [8]. In pharmacological experiments, insulin was shown to overcome scopolamine-induced memory impairment in rats trained in a radial arm maze task [4]. On the other hand the deterioration of CNS insulin receptor functions is associated with the pathogenesis of aging-related brain degenerative dementias such as sporadic Alzheimer’s disease [15–17,41,34]. Brain insulin and insulin receptor are reduced in the brains of sporadic Alzheimer’s disease patients [13]. Consistent results have also been found in rodents whose brains show aging-reduced insulin receptor numbers [46].
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Fig. 4. The effect of intra-CA1 daily microinjection of insulin on water maze performance. (A) The learning curves. (B) Average escape latency. (C) Average distance traveled. (D) The probe trial. Control-saline (A), control-0.5 insulin (F), control-6 insulin (G), control-12 insulin (H).
In our previous study we demonstrated that pre-training microinjection of insulin to CA1 region of the hippocampus can affect learning and memory in a dose-dependent manner. With lower dose (0.5 MU) insulin slightly impaired learning, with intermediate doses it had no significant effect on it and with higher doses it significantly improved spatial learning and memory. However the impairing effect of the lower dose was much weaker than the improving effects of the higher doses [27]. In conclusion, the current study examined the hypothesis that stress-induced deficit in spatial learning and memory can be prevented by intrahippocampal insulin injection. While the results from our behavioral experiments seem to implicate insulin as an influential factor in memory and hippocampal plasticity, much remains to be learned about the specific cellular and molecular mechanisms involved. These results demonstrate the effectiveness of insulin as a key neuro-protective agent. Acknowledgments This work was supported by a grant from Neuroscience Research Center of Shaheed Beheshti University of Medical Sciences. Help from Ms. Shiva for the preparation of the manuscript and Mr. Akbari for technical assistance are very much appreciated.
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