Modulation of synapse-related gene expression in the cerebellum and prefrontal cortex of rats subjected to the contextual fear conditioning paradigm.

The contextual fear conditioning (CFC) paradigm is the most productive approach for understanding the neurobiology of learning and memory as it allows to follow the evolution of memory traces of a conditioned stimulus and a specific context. The formation of long-term memory involves alterations in synaptic efficacy and neural transmission. It is known that the prefrontal cortex (PFC) exerts top-down control over subcortical structures to regulate behavioural responses. Moreover, cerebellar structures are involved in storing conditioned responses. The purpose of this research was to determine if the response to conditioning and stressful challenge is associated with alterations in synapse-related genes mRNA levels in the PFC, cerebellar vermis (V), and hemispheres (H) of young adult male rats. Four groups of Wistar rats were examined: naïve, CFC, shock only (SO), and exploration (EXPL). The behavioural response was evaluated by measuring the total freezing duration. Real-Time PCR was employed to quantify mRNA levels of some genes involved in synaptic plasticity. The results obtained from this study showed alterations in gene expression in different synapse-related genes after exposure to stressful stimuli and positioning to new environment. In conclusion, conditioning behavioural stimuli change the expression profile of molecules involved in neural transmission.

Histaminergic Neurotransmission as a Gateway for the Cognitive Effect of Oleoylethanolamide in Contextual Fear Conditioning.

Background: The integrity of the brain histaminergic system is necessary for the unfolding of homeostatic and cognitive processes through the recruitment of alternative circuits with distinct temporal patterns. We recently demonstrated that the fat-sensing lipid mediator oleoylethanolamide indirectly activates histaminergic neurons to exerts its hypophagic effects. The present experiments investigated whether histaminergic neurotransmission is necessary also for the modulation of emotional memory induced by oleoylethanolamide in a contextual fear conditioning paradigm. Methods: We examined the acute effect of i.p. administration of oleoylethanolamide immediately posttraining in the contextual fear conditioning test. Retention test was performed 72 hours after training. To test the participation of the brain histaminergic system in the cognitive effect of oleoylethanolamide, we depleted rats of brain histamine with an i.c.v. injection of alpha-fluoromethylhistidine (a suicide inhibitor of histidine decarboxylase) or bilateral intra-amygdala infusions of histamine H1 or H2 receptor antagonists. We also examined the effect of oleoylethanolamide on histamine release in the amygdala using in vivo microdialysis. Results: Posttraining administration of oleoylethanolamide enhanced freezing time at retention. This effect was blocked by both i.c.v. infusions of alpha-fluoromethylhistidine or by intra-amygdala infusions of either pyrilamine or zolantidine (H1 and H2 receptor antagonists, respectively). Microdialysis experiments showed that oleoylethanolamide increased histamine release from the amygdala of freely moving rats. Conclusions: Our results suggest that activation of the histaminergic system in the amygdala has a "permissive" role on the memory-enhancing effects of oleoylethanolamide. Hence, targeting the H1 and H2 receptors may modify the expression of emotional memory and reduce dysfunctional aversive memories as found in phobias and posttraumatic stress disorder.

Memory retrieval of inhibitory avoidance requires histamine H1 receptor activation in the hippocampus.

Retrieval represents a dynamic process that may require neuromodulatory signaling. Here, we report that the integrity of the brain histaminergic system is necessary for retrieval of inhibitory avoidance (IA) memory, because rats depleted of histamine through lateral ventricle injections of α-fluoromethylhistidine (a-FMHis), a suicide inhibitor of histidine decarboxylase, displayed impaired IA memory when tested 2 d after training. a-FMHis was administered 24 h after training, when IA memory trace was already formed. Infusion of histamine in hippocampal CA1 of brain histamine-depleted rats (hence, amnesic) 10 min before the retention test restored IA memory but was ineffective when given in the basolateral amygdala (BLA) or the ventral medial prefrontal cortex (vmPFC). Intra-CA1 injections of selective H1 and H2 receptor agonists showed that histamine exerted its effect by activating the H1 receptor. Noteworthy, the H1 receptor antagonist pyrilamine disrupted IA memory retrieval in rats, thus strongly supporting an active involvement of endogenous histamine; 90 min after the retention test, c-Fos-positive neurons were significantly fewer in the CA1s of a-FMHis-treated rats that displayed amnesia compared with in the control group. We also found reduced levels of phosphorylated cAMP-responsive element binding protein (pCREB) in the CA1s of a-FMHis-treated animals compared with in controls. Increases in pCREB levels are associated with retrieval of associated memories. Targeting the histaminergic system may modify the retrieval of emotional memory; hence, histaminergic ligands might reduce dysfunctional aversive memories and improve the efficacy of exposure psychotherapies.

Brain sites involved in fear memory reconsolidation and extinction of rodents.

Fear memory is a motivational system essential for organisms survival having a central role in organization of defensive behaviors to threat. In the last years there has been a growing interest on conditioned fear memory reconsolidation and extinction, two specific phases of memorization process, both induced by memory retrieval. Understanding the mechanisms underlying these two mnemonic processes may allow to work out therapeutic interventions for treatment of human fear and anxiety disorders, such as specific phobias and post-traumatic stress disorder. Based on the use of one-trial conditioning paradigms, which allow to follow the evolution of a mnemonic trace in its various phases, the present paper has attempted to reorganize the current literature relative to the rodents highlighting both the role of several brain structures in conditioned fear memory reconsolidation and extinction and the selective cellular processes involved. A crucial role seems to be play by medial prefrontal cortex, in particular by prelimbic and infralimbic cortices, and by distinct connections between them and the amygdala, hippocampus and entorhinal cortex.

Histamine in the basolateral amygdala promotes inhibitory avoidance learning independently of hippocampus.

Recent discoveries demonstrated that recruitment of alternative brain circuits permits compensation of memory impairments following damage to brain regions specialized in integrating and/or storing specific memories, including both dorsal hippocampus and basolateral amygdala (BLA). Here, we first report that the integrity of the brain histaminergic system is necessary for long-term, but not for short-term memory of step-down inhibitory avoidance (IA). Second, we found that phosphorylation of cyclic adenosine monophosphate (cAMP) responsive-element-binding protein, a crucial mediator in long-term memory formation, correlated anatomically and temporally with histamine-induced memory retrieval, showing the active involvement of histamine function in CA1 and BLA in different phases of memory consolidation. Third, we found that exogenous application of histamine in either hippocampal CA1 or BLA of brain histamine-depleted rats, hence amnesic, restored long-term memory; however, the time frame of memory rescue was different for the two brain structures, short lived (immediately posttraining) for BLA, long lasting (up to 6 h) for the CA1. Moreover, long-term memory was formed immediately after training restoring of histamine transmission only in the BLA. These findings reveal the essential role of histaminergic neurotransmission to provide the brain with the plasticity necessary to ensure memorization of emotionally salient events, through recruitment of alternative circuits. Hence, our findings indicate that the histaminergic system comprises parallel, coordinated pathways that provide compensatory plasticity when one brain structure is compromised.

Entorhinal cortex contribution to contextual fear conditioning extinction and reconsolidation in rats.

During contextual fear conditioning a rat learns a temporal contiguity association between the exposition to a previously neutral context (CS) and an aversive unconditioned stimulus (US) as a footshock. This condition determines in the rat the freezing reaction during the subsequent re-exposition to the context. Potentially the re-exposition without US presentation initiates two opposing and competing processes: reconsolidation and extinction. Reconsolidation process re-stabilizes and strengthens the original memory and it is initiated by a brief re-exposure to context. Instead the extinction process leads to the decrease of the expression of the original memory and it is triggered by prolonged re-exposure to the context. Here we analyzed the entorhinal cortex (ENT) participation in contextual fear conditioning reconsolidation and extinction. The rats were trained in contextual fear conditioning and 24h later they were subjected either to a brief (2 min) reactivation session or to a prolonged (120 min) re-exposition to context to induce extinction of the contextual fear memory. Immediately after the reactivation or the extinction session, the animals were submitted to bilateral ENT TTX inactivation. Memory retention was assessed as conditioned freezing duration measured 72 h after TTX administration. The results showed that ENT inactivation both after reactivation and extinction session was followed by contextual freezing retention impairment. Thus, the present findings point out that ENT is involved in contextual fear memory reconsolidation and extinction. This neural structure might be part of parallel circuits underlying two phases of contextual fear memory processing.

Modulation of gene expression in contextual fear conditioning in the rat.

In contextual fear conditioning (CFC) a single training leads to long-term memory of context-aversive electrical foot-shocks association. Mid-temporal regions of the brain of trained and naive rats were obtained 2 days after conditioning and screened by two-directional suppression subtractive hybridization. A pool of differentially expressed genes was identified and some of them were randomly selected and confirmed with qRT-PCR assay. These transcripts showed high homology for rat gene sequences coding for proteins involved in different cellular processes. The expression of the selected transcripts was also tested in rats which had freely explored the experimental apparatus (exploration) and in rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make the association between painful stimuli and the apparatus difficult (shock-only). Some genes resulted differentially expressed only in the rats subjected to CFC, others only in exploration or shock-only rats, whereas the gene coding for translocase of outer mitochondrial membrane 20 protein and nardilysin were differentially expressed in both CFC and exploration rats. For example, the expression of stathmin 1 whose transcripts resulted up regulated was also tested to evaluate the transduction and protein localization after conditioning.

Fimbria-fornix and entorhinal cortex differential contribution to contextual and cued fear conditioning consolidation in rats.

The Fimbria-Fornix (FF) and Entorhinal Cortex (EC) are the primary interfaces between the hippocampus and, respectively, subcortical structures and cortical areas. Their mnemonic role has been repeatedly proposed. In order to investigate their role in fear conditioning, FF and EC were subjected to bilateral fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats that had undergone training for fear conditioning to an acoustic stimulus (CS) and context. TTX was stereotaxically injected into animals of different groups at increasing post-acquisition delays. Memory was assessed as conditioned freezing duration measured during retention testing, performed 72 and 96 h after TTX administration in a counterbalanced manner. The results showed that FF inactivation, performed immediately after conditioning, did not disrupt consolidation of either contextual or auditory fear memory. On the contrary, EC inactivation performed at the same time was followed by both contextual and CS fear response retention impairment. EC inactivation performed 1.5h post-acquisition impaired only contextual fear response retention. EC inactivation performed 24h after acquisition training had no effect on the consolidation process. The present findings show a clearly different role of FF and EC in fear conditioning consolidation in the rat. The results are discussed in relation to their known connections with the hippocampus.

Histaminergic ligands injected into the nucleus basalis magnocellularis differentially affect fear conditioning consolidation.

The role of the nucleus basalis magnocellularis (NBM) in fear conditioning encoding is well established. In the present report, we investigate the involvement of the NBM histaminergic system in consolidating fear memories. The NBM was injected bilaterally with ligands of histaminergic receptors immediately after contextual fear conditioning. Histaminergic compounds, either alone or in combination, were stereotaxically administered to different groups of adult male Wistar rats and memory was assessed as conditioned freezing duration 72 h after administration. This protocol prevents interference with NBM function during either acquisition or retrieval phases, hence restricting the effect of pharmacological manipulations to fear memory consolidation. The results presented here demonstrate that post-training H3 receptors (H3R) blockade with the antagonist/inverse agonist thioperamide or activation with immepip in the NBM potentiates or decreases, respectively, freezing response at retrieval. Thioperamide induced memory enhancement seems to depend on H2R, but not H1R activation, as the H2R antagonist zolantidine blocked the effect of thioperamide, whereas the H1R antagonist pyrilamine was ineffective. Furthermore, the H2R agonist ampthamine improved fear memory expression independently of the H3R agonist effect. Our results indicate that activation of post-synaptic H2R within the NBM by endogenous histamine is responsible for the potentiated expression of fear responses. The results are discussed in terms of activation of H3 auto- and heteroreceptors within the NBM and the differential effect of H3R ligands on fear memory consolidation in distinct brain regions.

Maternal dietary loads of α-tocopherol differentially influence fear conditioning and spatial learning in adult offspring.

α-Tocopherol, the main component of vitamin E, is well known to be a radical scavenger, so an increased intake of vitamin E is recommended in complicated pregnancy, to prevent possible fetus damage by free radical. In a previous work, we found that maternal α-tocopherol supplementation affects PKC-mediated cellular signaling and hippocampal synaptic plasticity in developing brain; the latter effect persists in adulthood. Here, adult rats maternally exposed to supranutritional doses of α-tocopherol were evaluated for Contextual Fear Conditioning and spatial learning in Morris Water Maze, two different hippocampus-dependent learning tasks. Moreover, anxiety, spontaneous activity, and explorative drive were also evaluated as factors potentially affecting learning performance. Treated rats showed a different behavior with respect to controls: performance in Contextual Fear Conditioning was improved, while spatial learning tested in Morris Water Maze, was impaired. The improvement of fear response was not ascribable to differences in anxiety level and/or spontaneous activity; thus it appears to be a specific effect of α-tocopherol overloading during brain development. On the contrary, the impaired performance in Morris Water Maze exhibited by treated rats can be in part explained by their enhanced explorative drive. Although extrapolation from rats to humans is difficult, a caveat in assuming supranutritional doses of vitamin E in pregnancy arises from this study.

Maternal dietary loads of α-tocopherol depress protein kinase C signaling and synaptic plasticity in rat postnatal developing hippocampus and promote permanent deficits in adult offspring.

Vitamin E (α-tocopherol) supplementation has been tested as prophylaxis against gestational disorders associated with oxidative damage. However, recent evidence showing that high maternal α-tocopherol intake can adversely affect offspring development raises concerns on the safety of vitamin E extradosages during pregnancy. Besides acting as an antioxidant, α-tocopherol depresses cell proliferation and modulates cell signaling through inhibiting protein kinase C (PKC), a kinase that is deeply involved in neural maturation and plasticity. Possible effects of α-tocopherol loads in the maturing brain, where PKC dysregulation is associated to developmental dysfunctions, are poorly known. Here, supranutritional doses of α-tocopherol were fed to pregnant and lactating dams to evaluate the effects on PKC signaling and morphofunctional maturation in offspring hippocampus. Results showed that maternal supplementation potentiates hippocampal α-tocopherol incorporation in offspring and leads to marked decrease of PKC phosphorylation throughout postnatal maturation, accompanied by reduced phosphorylation of growth-associated protein-43 and myristoylated alanine-rich C kinase substrate, two PKC substrates involved in neural development and plasticity. Although processes of neuronal maturation, synapse formation and targeting appeared unaffected, offspring of supplemented mothers displayed a marked reduction of long-term synaptic plasticity in juvenile hippocampus. Interestingly, this impairment persisted in adulthood, when a deficit in hippocampus-dependent, long-lasting spatial memory was also revealed. In conclusion, maternal supplementation with elevated doses of α-tocopherol can influence cell signaling and synaptic plasticity in developing hippocampus and promotes permanent adverse effects in adult offspring. The present results emphasize the need to evaluate the safety of supranutritional maternal intake of α-tocopherol in humans.

Substantia nigra, nucleus basalis magnocellularis and basolateral amygdala roles in extinction of contextual fear conditioning in the rat.

Fear conditioning is accepted as a useful experimental paradigm to investigate anxious disorders following stress. In this field it is important to understand the mechanisms underlying the extinction of conditioned fear. In the rat it has been shown that the amygdalar basolateral nucleus plays a crucial role in all memorization phases of this type of memory (acquisition, consolidation, retrieval, and also reconsolidation and extinction). Recent results show that both the substantia nigra and nucleus basalis magnocellularis, two sites strongly connected with the basolateral amygdala are also involved in the consolidation of contextual fear conditioning. The aim of the present work is to investigate if latter two sites, besides the basolateral amygdala, are also involved in the extinction of the conditioned fear response. The results show that tetrodotoxin-induced inactivation of post-extinction training of either site does not impair the extinction process, which instead is impaired by inactivation of the basolateral amygdala. Thus, the present results confirm previous ones which show that diverse memorization phases (post-acquisition consolidation, extinction, reconsolidation) may be sustained by different neural sites and circuits.

Effects of dietary extra-virgin olive oil on behaviour and brain biochemical parameters in ageing rats.

The aim of the present study was to verify whether extra-virgin olive oil, a dietary component naturally containing phenolic antioxidants, has the potential to protect the brain from the deleterious effects of ageing. To accomplish this goal, we used male rats fed a high-energy diet containing either maize oil, or extra-virgin olive oil with high or low phenol content (720 or 10 mg total phenols/kg oil, corresponding to a daily dose of 4 or 0.05 mg total phenols/kg body weight, respectively) from age 12 months to senescence. The measured endpoints were biochemical parameters related to oxidative stress and functional tests to evaluate motor, cognitive and emotional behaviour. Olive oil phenols did not exert major protective actions on motor and cognitive function, as we observed only a tendency to improved motor coordination on the rotarod in the old animals treated with the oil rich in phenols (40 % average increase in the time to first fall; P = 0.18). However, an interesting finding of the present study was a reduced step-through latency in the light-dark box test, found in the older animals upon treatment with the oil rich in antioxidant phenols, possibly indicating an anxiety-lowering effect. This effect was associated with decreased glutathione reductase activity and expression in the brain, a phenomenon previously associated with decreased anxiety in rodents. These results indicate a previously undetected effect of a diet containing an olive oil rich in phenols. Further studies are warranted to verify whether specific food antioxidants might also have an effect on emotional behaviour.

Differential roles of the basolateral amygdala and nucleus basalis magnocellularis during post-reactivation contextual fear conditioning reconsolidation in rats.

The roles of the basolateral amygdala and nucleus basalis magnocellularis in fear conditioning reconsolidation were investigated by means of tetrodotoxin bilateral inactivation performed 96 h after conditioning, immediately after reactivation training. Footshocks of 1.2 mA intensity were employed to induce the generalization phenomenon. Basolateral amygdala inactivation disrupts the contextual fear response and its generalization but not acoustic CS trace retention, when measured 72 and 96 h after tetrodotoxin administration. Nucleus basalis magnocellularis functional inactivation does not affect memory post-reactivation phase of any of the three conditioned fear responses. The present findings show a differential role of the two structures in fear memory reconsolidation and can be a starting point for future investigation of the neural circuits subserving generalization.

The role of the nucleus basalis magnocellularis in fear conditioning consolidation in the rat.

The nucleus basalis magnocellularis (NBM) is known to be involved in the memorization of several conditioned responses. To investigate the role of the NBM in fear conditioning memorization, this neural site was subjected to fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats that had undergone fear training to acoustic conditioned stimulus (CS) and context. TTX was stereotaxically administered to different groups of rats at increasing intervals after the acquisition session. Memory was assessed as the conditioned freezing duration measured during retention testing, always performed 72 and 96 h after TTX administration. In this way, there was no interference with normal NBM function during either acquisition or retrieval phases, allowing any amnesic effect to be due only to consolidation disruption. The results show that for contextual fear response memory consolidation, NBM functional integrity is necessary up to 24 h post-acquisition. On the other hand, NBM functional integrity was shown to be necessary for memory consolidation of the acoustic CS fear response only immediately after acquisition and not 24-h post-acquisition. The present findings help to elucidate the role of the NBM in memory consolidation and better define the neural circuits involved in fear memories.

Histamine in the brain: beyond sleep and memory.

A few decades elapsed between the attribution of unwanted side effects of classic antihistamine compounds to the blockade of central H(1) receptors, and the acceptance of the concept that the histaminergic system commands general states of metabolism and consciousness. In the early 80s, two laboratories discovered independently that histaminergic neurons are located in the posterior hypothalamus and project to the whole CNS [Panula P, Yang HY, Costa E. Histamine-containing neurons in the rat hypothalamus. Proc Natl Acad Sci 1984;81:2572-76, Watanabe T, Taguchi Y, Hayashi H, Tanaka J, Shiosaka S, Tohyama M, Kubota H, Terano Y, Wada H. Evidence for the presence of a histaminergic neuron system in the rat brain: an immunohistochemical analysis. Neurosci Lett 1983;39:249-54], suggesting a global nature of histamine regulatory effects. Recently, functional studies demonstrated that activation of the central histaminergic system alters CNS functions in both behavioral and homeostatic contexts, which include sleep and wakefulness, learning and memory, anxiety, locomotion, feeding and drinking, and neuroendocrine regulation. These actions are achieved through interactions with other neurotransmitter systems, and the interplay between histaminergic neurons and other neurotransmitter systems are becoming clear. Hence, numerous laboratories are pursuing novel compounds targeting the three known histamine receptors found in the brain for various therapeutic indications. Preclinical studies are focusing on three major areas of interest and intense research is mainly oriented towards providing drugs for the treatment of sleep, cognitive and feeding disorders. This commentary is intended to summarize some of the latest findings that suggest functional roles for the interplay between histamine and other neurotransmitter systems, and to propose novel interactions as physiological substrates that may partially underlie some of the behavioral changes observed following manipulation of the histaminergic system.

Substantia nigra role in fear conditioning consolidation.

The substantia nigra (SN) is known to be involved in the memorization of several conditioned responses. To investigate the role of the SN in fear conditioning consolidation this neural site was subjected to fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats which had undergone fear training to acoustic CS and context. TTX was stereotaxically administered to different groups of rats at increasing intervals after the acquisition session. Memory was assessed as conditioned freezing duration measured during retention testing, always performed 72 and 96 h after TTX administration. In this way there was no interference with normal SN function during either acquisition or retrieval phases, so that any amnesic effect could be due only to consolidation disruption. The results show that SN functional integrity is necessary for contextual fear response consolidation up to the 24-h after-acquisition delay. On the contrary SN functional integrity was shown not to be necessary for the consolidation of acoustic CS fear responses. The present findings help to elucidate the role of the SN in memory consolidation and better define the neural circuits involved in fear memories.

Aversive memory reactivation engages in the amygdala only some neurotransmitters involved in consolidation.

Consolidation refers to item stabilization in long-term memory. Retrieval renders a consolidated memory sensitive, and a "reconsolidation" process has been hypothesized to keep the original memory persistent. Some authors could not detect this phenomenon. Here we show that retrieved contextual fear memory is vulnerable to amnesic treatments and that the amygdala is critically involved. Cholinergic and histaminergic systems seem to modulate only consolidation, whereas cannabinoids are involved in both consolidation and reactivation. The lability of retrieved memory affords opportunities to treat disorders such as phobias, post-traumatic stress, or chronic pain, and these results help searching for appropriate therapeutic targets.

Age-related naturally occurring depression of hippocampal neurogenesis does not affect trace fear conditioning.

New neuron production throughout adulthood in granule cell layer (GCL) of rat hippocampus is a well-known phenomenon. A role of new neurons in hippocampal learning has been proposed, but the question is still open. A reduction of neural precursor proliferation in GCL of 2-month-old rats to about 20%, induced by the cytostatic agent methylazoxymethanol, was found to cause impairment in trace conditioning, suggesting a role of immature neurons in this kind of hippocampus-dependent learning (Shors et al., Hippocampus 2002;12:578-584). Neurogenesis decreases with increasing age. In this study, neural precursor proliferation and newborn cell survival were evaluated in GCL of adult rats within a range of ages following development and preceding old age. In 5-month-old rats, neural precursor proliferation was reduced to 57% and newborn cell survival was reduced to 40% in comparison to rats of 2 months of age; in 12-month-old rats, the decrease was to 5 and 4%, respectively. Consistently, the density of immature neurons decreased to 41 and 13% in 5- and 12-month-old rats, respectively. The role of neurogenesis in trace fear conditioning was studied in this natural model of neurogenesis depression. No impairment in trace fear conditioning was found both in 5- and 12-month-old rats in comparison to 2-month-old rats, notwithstanding the decrease of neurogenesis that is marked in 12-month-old rats. This finding shows that a lower rate of neurogenesis is sufficient for learning in 12-month-old rats in comparison to young rats.

Spatial navigation in the Morris water maze: working and long lasting reference memories.

Spatial navigation development in the Morris water maze (MWM) paradigm was studied in 70-day-old male Long-Evans hooded rats. During 5 consecutive days, rats' training consisted of a daily block of 10 trials. Escape latency was measured in each trial. Probe testing was performed every day immediately before and after the daily block of trials. In addition, a final probe was performed on Day 6. During the first 3 days of training, the escape latency became progressively shorter, showing an asymptotic trend on Days 4 and 5. Probe trials administered at the end of the first acquisition sessions showed clear preference for the target quadrant but this information was not recalled at probe trials given 24h later. The memory trace retrieved after 24-h delay was formed only after 30 trials received over three sessions. The probe trial given at the end of an acquisition session tests the efficiency of the working memory whereas the 24h delayed probe trial reflects better-consolidated spatial information corresponding to long lasting reference memory. It can be noted that the progressive shortening of escape latencies does not express closely the evolution of the rat's long lasting (consolidated) reference memory. This memory can be satisfactorily measured only by probe testing performed at an adequate delay after training. These considerations may be of some interest when interpreting the rat's performance in the MWM.