Learning associated increase in heat shock cognate 70 mRNA and protein expression

The Morris water maze is a task widely used to investigate cellular and molecular changes associated with spatial learning and memory. This task has both spatial and aversive (swimming related stress) components. It is possible that stress may influence cellular modifications observed after learning the Morris water maze spatial task. Heat shock proteins, also known as stress proteins, are up-regulated in response to thermal stress, trauma, or environmental insults. In the rat hippocampus, psychophysiological stress increases the levels of heat shock protein 70 (HSC70). In this study, we investigated whether the expression of the hsc70 gene is modulated in the hippocampus during learning of the Morris water maze task. Five groups of rats were trained in the Morris water maze task for varying amounts of time (either 1, 2, 3, 4, or 5 days). Training consisted of 10 trials/day in which the animals were given 60s to find a submerged platform. Rats were sacrificed 24h after their last training trial. Results showed a significant increase in hsc70 mRNA and protein levels in the hippocampal formation after two and three days of training, respectively. The increase in mRNA and protein was associated with learning but not stress because the increase was not observed in the yoked control animals. These findings suggest that cellular and molecular changes can occur independent of stress. Moreover, the results are the first to implicate hsc70 expression in spatial learning.     

Hippocampal expression of the orphan nuclear receptor gene hzf-3/nurr1 during spatial discrimination learning

The immediate-early gene hzf-3, also known as nurr1, is a member of the inducible orphan nuclear receptor family and is one candidate in the search for genes associated with learning and memory processes. Here we report that acquisition of a spatial food search task is accompanied by elevated levels of hzf-3 mRNA in the hippocampus. Adult male Long-Evans rats were handled, food-restricted, and allowed to habituate to the maze prior to training. During acquisition, rats were given one training session per day for 5 days. Each training session consisted of five trials in which animals searched the maze for food located in 4 of 16 holes in the floor of the maze. Training resulted in spatial acquisition of the task. Northern blot analysis showed significant increases in hippocampal hzf-3 mRNA 3 h after training in the maze. Next, brains were obtained from Naive, Habituated, Day 1, Day 3, and Day 5 animals and processed for in situ hybridization. The results showed significant increases of hzf-3 mRNA in CA1 and CA3 subregions of the dorsal hippocampus during acquisition of the task. We conclude that expression of the hzf-3 gene in the brain is associated with long-term spatial memory processes. The present results are the first to implicate an orphan nuclear receptor in long-term information storage in the hippocampus.     

Working memory task decreases the survival of newly born neurons in hippocampus

Throughout life new neurons are generated in dentate gyrus of hippocampus. Previous studies have found that spatial tasks can rescue newly born neurons from death. However, it is still unknown whether new neurons are similarly affected by all types of hippocampal-dependent tasks. Here we investigated the possible effects of working memory task (WMT) on immature neurons. Mice were trained in reference memory task and WMT respectively. The reference memory task used the classical hidden platform (HP) water maze task, while WMT used a delayed matching-to-place (DMTP) water maze task. Bromodeoxyuridine (BrdU) was administrated during the early or late phase of training, or 1week prior to training, in order to label dividing proliferating cells. After water maze training, the number of BrdU-labeled cells in dentate gyrus of hippocampus was compared. In addition, hippocampal brain-derived neurotrophic factor (BDNF) and Notch 1 receptor were characterized using Western blot. Serum corticosterone levels were also measured using enzyme immunoassay. Results showed that HP task and DMTP task did not change the number of BrdU-labeled cells produced during the early or late phase of training. As expected, the HP task increased the number of BrdU-labeled cells produced 1 week prior to training. However, DMTP task decreased the number of BrdU-labeled cells produced 1 week prior to training. Both tasks lead to a significant increase in serum corticosterone levels and did not change the expression of BDNF and Notch 1 receptor in hippocampus. Taken together, these results demonstrate that WMT has different effects on survival of immature neurons, and therefore suggests immature neurons may have more than one role depending on the demands of the tasks.     

Individual differences in anxiety trait are related to spatial learning abilities and hippocampal expression of mineralocorticoid receptors

Although high levels of anxiety might be expected to negatively influence learning and memory, it remains to be shown whether individual differences in anxiety may influence spatial learning and memory in outbred rat populations. We have studied this possibility in male Wistar rats whose levels of anxiety were first characterized as either high (HA) or low (LA) according to their behavior in the elevated plus maze or in the open field test. Subsequently, their performance in the Morris water maze was studied, a task dependent on hippocampal activity. Interestingly, LA rats showed a faster acquisition and better memory in the water maze when compared to HA rats. Indeed, this difference in performance could mainly be attributed to the increase in thigmotactic behavior (swimming in circles close to the maze walls) displayed by HA rats during spatial navigation. Glucocorticoids are known to affect the state of anxiety and the hippocampus is the main target of glucocorticoids in the brain. Hence, we investigated whether the hippocampal expression of the two classical corticosteroid receptors, mineralocorticoid (MR) and glucocorticoid (GR) differed in the two groups of rats. We found that LA rats displayed higher hippocampal expression of MR but not GR than HA rats. Indeed, the expression levels for these receptors were positively correlated with the amount of time spent by the animals in the open arms of the elevated plus maze. Moreover, we present evidence that the levels of anxiety quantified in the first stages of our study constitute a trait rather than a state. Taken together, this study has generated evidence of a close interaction between the anxiety trait, hippocampal MR expression and the learning abilities of individuals in stressful spatial orientation tasks.     

Effect of neonatal handling on adult rat spatial learning and memory following acute stress

Brief neonatal handling permanently alters hypothalamic-pituitary-adrenal axis function resulting in increased ability to cope with stress. Since stress is known to affect cognitive abilities, in the present study we investigated the effect of brief (15 min) handling on learning and memory in the Morris water maze, following exposure to an acute restraint stress either before training or recall. Exposure of non-handled rats to the acute stress prior to training resulted in quicker learning of the task, than in the absence of the stressor. When acute stress preceded acquisition, male handled rats showed an overall better learning performance, and both sexes of handled animals were less impaired in the subsequent memory trial, compared to the respective non-handled. In addition, the number of neurons immunoreactive for GR was higher in all areas of Ammon's horn of the handled rats during the recall. In contrast, the number of neurons immunoreactive for MR was higher in the CA1 and CA2 areas of the non-handled males. When the acute restraint stress was applied prior to the memory test, neonatal handling was not effective in preventing mnemonic impairment, as all animal groups showed a similar deficit in recall. In this case, no difference between handled and non-handled rats was observed in the number of GR positive neurons in the CA2 and CA3 hippocampal areas during the memory test. These results indicate that early experience interacts with sex and acute stress exposure in adulthood to affect performance in the water maze. Hippocampal corticosterone receptors may play a role in determining the final outcome.     

Spatial navigation in complex and radial mazes in APP23 animals and neurotrophin signaling as a biological marker of early impairment

Impairment of hippocampal function precedes frontal and parietal cortex impairment in human Alzheimer's disease (AD). Neurotrophins are critical for behavioral performance and neuronal survival in AD. We used complex and radial mazes to assess spatial orientation and learning in wild-type and B6-Tg(ThylAPP)23Sdz (APP23) animals, a transgenic mouse model of AD. We also assessed brain content of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). Performance was alike in wild-type and APP23 animals in the radial maze. In contrast, performance in the complex maze was better in wild-type than APP23 animals. Contrary to the wild-type, hippocampal BDNF levels decreased on training in APP23 animals. Hippocampal and frontal cortex NGF levels in APP23 animals correlated with the time to solve the complex maze, but correlated inversely with escape time in wild-type animals. NT-3 levels were alike in wild-type and APP23 animals and were unchanged even after training. Both types of mazes depend on hippocampal integrity to some extent. However, according to the cognitive mapping theory of spatial learning, the complex maze because of the increased complexity of the environment most likely depends more strongly on preserved hippocampal function than the radial maze in the working memory configuration applied here. Greater impairment in complex maze performance than in radial maze performance thus resembles the predominant affliction of the loss of hippocampal function in human AD. NGF and BDNF levels on maze learning are different in wild-type and transgenic animals, indicating that biological markers of AD may be altered on challenge even though equilibrium levels are alike.     

Memory-enhancing corticosterone treatment increases amygdala norepinephrine and Arc protein expression in hippocampal synaptic fractions

Considerable evidence indicates that glucocorticoid hormones enhance the consolidation of memory for emotionally arousing events through interactions with the noradrenergic system of the basolateral complex of the amygdala (BLA). We previously reported that intra-BLA administration of a β-adrenoceptor agonist immediately after inhibitory avoidance training enhanced memory consolidation and increased hippocampal expression of the protein product of the immediate early gene activity-regulated cytoskeletal-associated protein (Arc). In the present experiments corticosterone (3 mg/kg, i.p.) was administered to male Sprague-Dawley rats immediately after inhibitory avoidance training to examine effects on long-term memory, amygdala norepinephrine levels, and hippocampal Arc expression. Corticosterone increased amygdala norepinephrine levels 15 min after inhibitory avoidance training, as assessed by in vivo microdialysis, and enhanced memory tested at 48 h. Corticosterone treatment also increased expression of Arc protein in hippocampal synaptic tissue. The elevation in BLA norepinephrine appears to participate in corticosterone-influenced modulation of hippocampal Arc expression as intra-BLA blockade of β-adrenoceptors with propranolol (0.5 μg/0.2 μL) attenuated the corticosterone-induced synaptic Arc expression in the hippocampus. These findings indicate that noradrenergic activity at BLA β-adrenoceptors is involved in corticosterone-induced enhancement of memory consolidation and expression of the synaptic-plasticity-related protein Arc in the hippocampus.     

Modulation of behavioral inhibition in appetitive extinction following manipulation of adrenal steroids in rats: implications for involvement of the hippocampus.

Corticosterone, the principal glucocorticoid in the rat, binds selectively to the CA1 pyramidal neurons of the hippocampus where the hormone has been demonstrated to exert a moderate chronic suppression of spontaneous activity. In the first experiment of the current study, the functional behavioral significance of this hormone--brain interaction was investigated in the extinction of an appetitive runway response in normal rats and those with lesions of the hippocampus. During extinction, half of the animals in each group were given daily subcutaneous injections of corticosterone. Whie the classical retardation effect of hippocampal lesions on appetitive extinction was replicated, hormone treatment was without effect in normal or hippocampally damaged subjects. The absence of a hormone effect in normals was primarily attributed to a saturated limited-binding system operating in the normal animal. Experiment 2 tested this notion, repeating the first experiment, with adrenal-ectomized (ADX), ADX + corticosterone replacement, and normal groups of animals. Adrenalectomy produced a striking facilitation of extinction which was speculated to be the result of a hyperactive inhibitory neural organ free from an inhibitory endocrine feedback. Corticosterone treatment normalized the progress of extinction in ADX animals, providing support for the afore-mentioned speculation. In the normal animal, it appears that a stress-induced surge in hormone level interacts with a limited-capacity neural binding to produce a transient dynamic range of behavioral disinhibition, perhaps promoting persistence during initial stages of frustrative nonreward in moderate stress tasks.     

Inhibitory avoidance deficit following short-term adrenalectomy in the rat: the role of adrenal catecholamines

Impaired retention of an inhibitory avoidance response was observed in rats subjected to adrenalectomy (ADX) up to 120 hr before the single learning trial. Corticosterone substitution failed to normalize this behavioral deficit. Rats ADX 240 hr prior to the learning trial showed a normalized behavior. Adrenomedullectomy (ADXM) 48 or 240 hr before learning caused a similar impairment as in short-term ADX rats. The 240-hr ADX rats subjected to corticosterone substitution showed the same behavioral deficit as short-term ADX rats or ADXM ones. Immediate postlearning subcutaneous injection of adrenaline in a dose range of 0.005-5.0 micrograms/kg or of noradrenaline (0.005-0.5 microgram/kg) to 48-hr ADX rats resulted in a dose-related improvement of later retention behavior. Higher doses of catecholamines were less or ineffective. Postlearning treatment of 48-hr ADXM rats with adrenaline (0.5-500 micrograms/kg) caused a similar pattern of behavioral changes. It is concluded that adrenal catecholamines play an important role in the modulation of consolidation of memory. In addition, the high circulating ACTH levels that follow long-term ADX may correct for the behaviorial deficit induced by the absence of adrenomedullary catecholamines.     

Retrieval induces hippocampal-dependent reconsolidation of spatial memory

Nonreinforced retrieval can cause extinction and/or reconsolidation, two processes that affect subsequent retrieval in opposite ways. Using the Morris water maze task we show that, in the rat, repeated nonreinforced expression of spatial memory causes extinction, which is unaffected by inhibition of protein synthesis within the CA1 region of the dorsal hippocampus. However, if the number of nonreinforced retrieval trials is insufficient to induce long-lasting extinction, then a hippocampal protein synthesis-dependent reconsolidation process recovers the original memory. Inhibition of hippocampal protein synthesis after reversal learning sessions impairs retention of the reversed preference and blocks persistence of the original one, suggesting that reversal learning involves reconsolidation rather than extinction of the original memory. Our results suggest the existence of a hippocampal protein synthesis-dependent reconsolidation process that operates to recover or update retrieval-weakened memories from incomplete extinction.     

Sleep deprivation selectively impairs memory consolidation for contextual fear conditioning

Many behavioral and electrophysiological studies in animals and humans have suggested that sleep and circadian rhythms influence memory consolidation. In rodents, hippocampus-dependent memory may be particularly sensitive to sleep deprivation after training, as spatial memory in the Morris water maze is impaired by rapid eye movement sleep deprivation following training. Spatial learning in the Morris water maze, however, requires multiple training trials and performance, as measured by time to reach the hidden platform is influenced by not only spatial learning but also procedural learning. To determine if sleep is important for the consolidation of a single-trial, hippocampus-dependent task, we sleep deprived animals for 0–5 and 5–10 h after training for contextual and cued fear conditioning. We found that sleep deprivation from 0–5 h after training for this task impaired memory consolidation for contextual fear conditioning whereas sleep deprivation from 5–10 h after training had no effect. Sleep deprivation at either time point had no effect on cued fear conditioning, a hippocampus-independent task. Previous studies have determined that memory consolidation for fear conditioning is impaired when protein kinase A and protein synthesis inhibitors are administered at the same time as when sleep deprivation is effective, suggesting that sleep deprivation may act by modifying these molecular mechanisms of memory storage.     

A Necessity for MAP Kinase Activation in Mammalian Spatial Learning

Although the biochemical mechanisms underlying learning and memory have not yet been fully elucidated, mounting evidence suggests that activation of protein kinases and phosphorylation of their downstream effectors plays a major role. Recent findings in our laboratory have shown a requirement for the mitogen-activated protein kinase (MAPK) cascade in hippocampal synaptic plasticity. Therefore, we used an inhibitor of MAPK activation, SL327, to test the role of the MAPK cascade in hippocampus-dependent learning in mice. SL327, which crosses the blood-brain barrier, was administered intraperitoneally at several concentrations to animals prior to cue and contextual fear conditioning. Administration of SL327 completely blocked contextual fear conditioning and significantly attenuated cue learning when measured 24 hr after training. To determine whether MAPK activation is required for spatial learning, we administered SL327 to mice prior to training in the Morris water maze. Animals treated with SL327 exhibited significant attenuation of water maze learning; they took significantly longer to find a hidden platform compared with vehicle-treated controls and also failed to use a selective search strategy during subsequent probe trials in which the platform was removed. These impairments cannot be attributed to nonspecific effects of the drug during the training phase; no deficit was seen in the visible platform task, and injection of SL327 following training produced no effect on the performance of these mice in the hidden platform task. These findings indicate that the MAPK cascade is required for spatial and contextual learning in mice.     

Preserved learning about allocentric cues but impaired flexible memory expression in rats with hippocampal lesions

Several studies have shown that slight modifications in the standard reference spatial memory procedure normally used for allocentric learning in the Morris water maze and the radial maze, can overcome the classic deficit in allocentric navigation typically observed in rats with hippocampal damage. In these special paradigms, however, there is only intramaze manipulation of a salient stimulus. The present study was designed to investigate whether extramaze manipulations produce a similar outcome. With this aim a four-arm plus-shaped maze and a reference spatial memory paradigm were used, in which the goal arm was marked in two ways: by a prominent extramaze cue (intermittent light), which maintained a constant relation with the goal, and by the extramaze constellation of stimuli around the maze. Experiment 1 showed that, unlike the standard version of the task, using this special training procedure hippocampally-damaged rats could learn a place response as quickly as control animals; importantly, one day after reaching criterion, lesioned and control subjects performed the task perfectly during a transfer test in which the salient extramaze stimulus used during the acquisition was removed. However, although acquisition deficit was overcomed in these lesioned animals, a profound deficit in retention was detected 15 days later. Experiment 2 suggests that although under our special paradigm hippocampal rats can learn a place response, spatial memory only can be expressed when the requisites of behavioral flexibility are minimal. These findings suggest that, under certain circumstances, extrahippocampal structures are sufficient for building a coherent allocentric representation of space; however, flexible memory expression is dependent, fundamentally, on hippocampal functioning.     

A facilitative role for corticosterone in the acquisition of a spatial task under moderate stress

Emotionally charged experiences alter memory storage via the activation of hormonal systems. Previously, we have shown that compared with rats trained for a massed spatial learning task in the water maze in warm water (25°C), animals that were trained in cold water (19°C) performed better and showed higher levels of the stress hormone corticosterone. Here, we examined whether manipulating the levels of corticosterone can determine the strength of spatial information acquisition and retention. Rats were injected with metyrapone (25, 50, and 75 mg/kg, i.p.) or with corticosterone (10 and 25 mg/kg, i.p.) and trained in a massed spatial task in either cold (19°C) or warm (25°C) water. We found that whereas animals injected with vehicle performed well in the spatial task in cold water (moderate stress), rats injected with the intermediate metyrapone dose showed impairment in performance. Moreover, whereas animals injected with vehicle on average did not perform well in warm water (mild stress), rats injected with the lower corticosterone dose showed improvement in performance in warm water. These two mirror experiments of corticosterone blockade and enhancement strongly suggest that corticosterone is instrumental in the acquisition and retention of the spatial learning task.     

A systemically administered beta-adrenoceptor antagonist blocks corticosterone-induced impairment of contextual memory retrieval in rats

Several studies have reported that glucocorticoids impair memory retrieval. The present study examined in male Sprague-Dawley rats the effects of systemically administered corticosterone on retrieval of memory for inhibitory avoidance training. Corticosterone (3.0mg/kg, s.c.) injected 30min before retention testing, 48h after training, significantly impaired retention performance, as compared to vehicle treatment, of rats tested in the training context. In contrast, corticosterone administration did not impair retrieval when rats were tested for retention in a different context. Corticosterone did also not impair retention performance of rats given a mild-intensity footshock that resulted in only weak, non-contextual memory. These findings strongly suggest that corticosterone selectively impaired retrieval of contextual information associated with the training context. The centrally acting beta-adrenoceptor antagonist propranolol (2.0mg/kg), co-administered in a dose that did not affect retention performance alone, blocked the impairment in contextual memory retrieval induced by corticosterone. These findings provide evidence for the view that glucocorticoids interact with noradrenergic mechanisms in influencing memory retrieval.