Characterizing age-related decline of recognition memory and brain activation profile in mice.

Episodic memory decline is one of the earlier deficits occurring during normal aging in humans. The question of spatial versus non-spatial sensitivity to age-related memory decline is of importance for a full understanding of these changes. Here, we characterized the effect of normal aging on both non-spatial (object) and spatial (object location) memory performances as well as on associated neuronal activation in mice. Novel-object (NOR) and object-location (OLR) recognition tests, respectively assessing the identity and spatial features of object memory, were examined at different ages. We show that memory performances in both tests were altered by aging as early as 15 months of age: NOR memory was partially impaired whereas OLR memory was found to be fully disrupted at 15 months of age. Brain activation profiles were assessed for both tests using immunohistochemical detection of c-Fos (neuronal activation marker) in 3and 15 month-old mice. Normal performances in NOR task by 3 month-old mice were associated to an activation of the hippocampus and a trend towards an activation in the perirhinal cortex, in a way that did significantly differ with 15 month-old mice. During OLR task, brain activation took place in the hippocampus in 3 month-old but not significantly in 15 month-old mice, which were fully impaired at this task. These differential alterations of the object- and object-location recognition memory may be linked to differential alteration of the neuronal networks supporting these tasks.

Long-Lasting Effects of Chronic Intermittent Alcohol Exposure in Adolescent Mice on Object Recognition and Hippocampal Neuronal Activity.

BACKGROUND: Binge drinking is popular and highly prevalent in teenagers. However, the long-term cognitive and neurobiological consequences of such practices are not yet fully understood. In this context, we therefore assessed in mice whether a chronic intermittent alcohol (CIA) exposure in adolescence had long-term consequences on object discrimination and memory performances, emotional behaviors, brain activity, and morphology. METHODS: C57BL/6JRj mice were treated with either saline or ethanol (EtOH) (2 g/kg/d, i.p., from postnatal days [PND] 30 to PND 44 every other day). The day following the last administration or later in adulthood (PND 71) mice were tested for different behavioral tests (novel object recognition, spontaneous alternation, light-dark box, elevated plus-maze, actimeter test), to assess object recognition, working memory performances, anxiety-like behavior, and locomotor activity. We also investigated neuronal activation of hippocampus, prefrontal and perirhinal cortices, and anatomical changes using immediate-early gene expression and longitudinal brain magnetic resonance imaging. RESULTS: Our results showed that adolescent mice exposed to CIA present a critical and persistent impairment of short-term object recognition performances. By contrast, spatial working memory was not impaired, nor was anxiety-like behavior. This altered object discrimination was associated with a biphasic change in neuronal activity in the hippocampus but without morphological changes. Indeed, c-Fos expression was specifically increased in the dorsal dentate gyrus (DG) of the hippocampus after the binge exposure, but then became significantly lower in adulthood both in the DG and the CA1 part of the hippocampus compared with adult saline pretreated mice. CONCLUSIONS: These findings provide evidence for adolescent vulnerability to the effects of intermittent binge EtOH exposure on object discrimination and hippocampal activity with long-lasting consequences.

Effects of acute administration of melatonin on attentional, executive, and working memory processes in rats.

Melatonin is a potential candidate for additive therapy in cancer, neurodegenerative, and mental disorders requiring administration during the activity phase. Nevertheless, because melatonin has mostly been used as a hypnotic, less is known about its cognitive effects. In this study, we investigated the effects of acute administration of melatonin on executive, attentional, and working memory processes in rats during the activity phase. Three doses of melatonin (6, 18, or 36 mg/kg) were tested and compared to a saline control group in two behavioral tests: the Attentional Set Shifting task (for attentional and executive processes assessment) and the Spontaneous Alternation test in a Y-maze (for working memory assessment). Our results revealed that, up to 36 mg/kg, the acute administration dose of melatonin did not alter the attentional or executive processes, nor the working memory in rats. Consequently, this result may be encouraging for the use of melatonin in additive therapy during the activity phase.

Design of donecopride, a dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer's disease treatment.

RS67333 is a partial serotonin subtype 4 receptor (5-HT4R) agonist that has been widely studied for its procognitive effect. More recently, it has been shown that its ability to promote the nonamyloidogenic cleavage of the precursor of the neurotoxic amyloid-ß peptide leads to the secretion of the neurotrophic protein sAPPα. This effect has generated great interest in RS67333 as a potential treatment for Alzheimer's disease (AD). We show herein that RS67333 is also a submicromolar acetylcholinesterase (AChE) inhibitor and therefore, could contribute, through this effect, to the restoration of the cholinergic neurotransmission that becomes altered in AD. We planned to pharmacomodulate RS67333 to enhance its AChE inhibitory activity to take advantage of this pleiotropic pharmacological profile in the design of a novel multitarget-directed ligand that is able to exert not only a symptomatic but also, a disease-modifying effect against AD. These efforts allowed us to select donecopride as a valuable dual (h)5-HT4R partial agonist (Ki = 10.4 nM; 48.3% of control agonist response)/(h)AChEI (IC50 = 16 nM) that further promotes sAPPα release (EC50 = 11.3 nM). Donecopride, as a druggable lead, was assessed for its in vivo procognitive effects (0.1, 0.3, 1, and 3 mg/kg) with an improvement of memory performances observed at 0.3 and 1 mg/kg on the object recognition test. On the basis of these in vitro and in vivo activities, donecopride seems to be a promising drug candidate for AD treatment.

Dual histamine H3R/serotonin 5-HT4R ligands with antiamnesic properties: pharmacophore-based virtual screening and polypharmacology.

In recent years, preclinical and clinical studies have generated considerable interest in the development of histamine H3 receptor (H3R) antagonists as novel treatment for degenerative disorders associated with impaired cholinergic function. To identify novel scaffolds for H3R antagonism, a common feature-based pharmacophore model was developed and used to screen the 17,194 compounds of the CERMN (Centre d'Etudes et de Recherche sur le Médicament de Normandie) chemical library. Out of 268 virtual hits which have been gathered in 34 clusters, we were particularly interested in tricyclic derivatives also exhibiting a potent 5HT4R affinity. Benzo[h][1,6]naphthyridine derivatives showed the highest H3R affinity, and compound 17 (H3R Ki = 41.6 nM; 5-HT4R Ki = 208 nM) completely reversed the amnesiant effect of scopolamine at 3 mg/kg in a spatial working memory experiment. For the first time we demonstrated the feasibility to combine H3R and 5-HT4R activities in a single molecule, raising the exciting possibility that dual H3R antagonist/5HT4R agonist have potential for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Genomic transcriptional profiling in LOU/C/Jall rats identifies genes for successful aging.

Inbred LOU/C/Jall rats are currently described as a model of successful aging. These rats have a longer healthy median lifespan than many other strains, do not develop obesity, diabetes, or tumor and more importantly they do not show cognitive decline with aging. This is the first study to examine gene expression changes in the inbred LOU/C/Jall rat hippocampus and frontal cortex. Microarray data from animals aged 5 and 26 months were compared to that obtained from the classical Wistar rat strain to potentially identify only the genes associated with successful aging. We have thus identified a set of 15 genes in the hippocampus and 70 genes in the frontal cortex that could be grouped into several clearly delineated clusters of highly correlated genes associated with a diversity of biological processes, including regulation of plasticity, inflammatory response, metabolic, catabolic and homeostatic processes, and transcription. Such a multiplicity of gene networks and diversity of biological functions were not observed in the Wistar rat strain. The gene expression profiles identified in aged the LOU/C/Jall rats' hippocampus and frontal cortex should be related to their intact cognitive abilities, such as those assessed through spontaneous alternation.

Environmental enrichment enhances episodic-like memory in association with a modified neuronal activation profile in adult mice.

Although environmental enrichment is well known to improve learning and memory in rodents, the underlying neuronal networks' plasticity remains poorly described. Modifications of the brain activation pattern by enriched condition (EC), especially in the frontal cortex and the baso-lateral amygdala, have been reported during an aversive memory task in rodents. The aims of our study were to examine 1) whether EC modulates episodic-like memory in an object recognition task and 2) whether EC modulates the task-induced neuronal networks. To this end, adult male mice were housed either in standard condition (SC) or in EC for three weeks before behavioral experiments (n = 12/group). Memory performances were examined in an object recognition task performed in a Y-maze with a 2-hour or 24-hour delay between presentation and test (inter-session intervals, ISI). To characterize the mechanisms underlying the promnesiant effect of EC, the brain activation profile was assessed after either the presentation or the test sessions using immunohistochemical techniques with c-Fos as a neuronal activation marker. EC did not modulate memory performances after a 2 h-ISI, but extended object recognition memory to a 24 h-ISI. In contrast, SC mice did not discriminate the novel object at this ISI. Compared to SC mice, no activation related to the presentation session was found in selected brain regions of EC mice (in particular, no effect was found in the hippocampus and the perirhinal cortex and a reduced activation was found in the baso-lateral amygdala). On the other hand, an activation of the hippocampus and the infralimbic cortex was observed after the test session for EC, but not SC mice. These results suggest that the persistence of object recognition memory in EC could be related to a reorganization of neuronal networks occurring as early as the memory encoding.

A biphasic and brain-region selective down-regulation of cyclic adenosine monophosphate concentrations supports object recognition in the rat.

BACKGROUND: We aimed to further understand the relationship between cAMP concentration and mnesic performance. METHODS AND FINDINGS: Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p.), rolipram (PDE4 inhibitor, 0.3 mg/kg, i.p.) and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p.) before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices) before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold), prefrontal (1.3 fold) and perirhinal (1.3 fold) cortices from controls rat while decreased in prefrontal cortex (∼0.83 to 0.62 fold) from drug-treated rats (except for milrinone+RS 67333 treatment). After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold) and the perirhinal cortex (2.1 fold) from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold) and milrinone (respectively 1.46 fold and 1.56 fold)-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h). CONCLUSIONS: Our results strongly suggest that a "pre-sample" early increase in cAMP levels followed by a specific lowering of cAMP concentrations in each brain sub-region linked to the object recognition paradigm support learning efficacy after a middle-term delay.

Repeated mild hypoxic exposures decrease anxiety-like behavior in the adult mouse together with an increased brain adrenomedullin gene expression.

Whereas severe hypoxia is known to contribute to neuronal death and to lead to neurological disturbances, mild hypoxia can also induce beneficial effects through endogenous adaptive responses. The aim of this study was to investigate the effects of mild hypoxia (8% O(2)) on cognitive and emotional behavior in the adult mouse. To this end, mice were submitted to repeated mild hypoxia exposure or normoxia during 6 weeks and underwent behavioral testing during the last 3 weeks. Hypoxia decreased anxiety-like behavior in the light/dark transition test, enhanced, albeit modestly, non-spatial recognition memory, but did not alter spontaneous locomotor activity, nor spatial learning. On additional mice, whole brain adrenomedullin mRNA expression was found to be increased at D1, D25 and D41 after hypoxia initiation and vascular endothelial growth factor (VEGF) mRNA expression was increased at only on D41. This work shows that repeated mild hypoxic exposure decreases anxiety-related behavior and points out hypoxia inducible factor-1 (HIF-1) target genes, particularly adrenomedullin, as potential mediator candidate.

Behavioral deficits after distal focal cerebral ischemia in mice: Usefulness of adhesive removal test.

Distal occlusion of the middle cerebral artery (dMCAo), which closely mimics human stroke, is one of the most used animal models. However, although assessment of histological and functional outcome is increasingly recommended for preclinical studies, the latter is often excluded because of the high difficulties to estimate, especially in mice, behavioral impairments. The aim of our study was to deeply screen functional consequences of distal permanent MCAo in mice to target relevant behaviors for future studies. A set of sensorimotor and cognitive tests were performed during 3 weeks postsurgery in 2 groups of mice. Afterward, brain infarctions were estimated by histological staining or magnetic resonance imaging. Overall, while no long-term functional impairments could be detected, the adhesive removal was the only test showing a deficit. Interestingly, this sensorimotor impairment was correlated to cortical damage 3 weeks after surgery. In conclusion, despite the fact that dMCAo-induced deficits could not be evidenced by most of our behavioral tests, the authors showed that the adhesive removal test was the only one, sensitive enough, to highlight a long-term deficit. This result suggests therefore that this mouse model of ischemia is relevant to efficiently assess therapeutic strategies with histological but also behavioral analysis, provided that relevant tests are used.

Increased particulate phosphodiesterase 4 in the prefrontal cortex supports 5-HT4 receptor-induced improvement of object recognition memory in the rat.

RATIONALE: Serotonin receptors (5-HT4Rs) are critical to both short-term and long-term memory processes. These receptors mainly trigger the cyclic adenosine monophosphate (cAMP)/protein kinase A signaling pathway, which is regulated by cAMP phosphodiesterases (PDEs). OBJECTIVES: We investigated the mechanisms underlying the effect of the selective activation of 5-HT4R on information acquisition in an object recognition memory task and the putative regulation of PDE. MATERIALS AND METHODS: The effect of RS 67333 (1 mg/kg, intraperitoneally [i.p.], injected 30 min before the sample phase) was examined at different delay intervals in an object recognition task in Sprague-Dawley rats. After the testing trial, PDE activity of brain regions implicated in this task was assayed. RESULTS: RS 67333-treated rats spent more time exploring the novel object after a 15-min (P < 0.001) or 4-h delay (P < 0.01) but not after a 24-h delay, whereas control animals showed no preference for the novel object for delays greater than 15 min. We characterized the specific patterns and kinetic properties of PDE in the prefrontal and perirhinal cortices as well as in the hippocampus. We demonstrated that particulate PDE activities increase in both the prefrontal cortex and hippocampus following 5-HT4R stimulation. In the prefrontal cortex, PDE4 activities support the RS 67333-induced modification of PDE activities, whereas in the hippocampus, all cAMP-PDE activities varied. In contrast, particulate PDE variation in the hippocampus was not found to support improvement of recognition memory after a 4-h delay. CONCLUSIONS: We provide evidence that the increase in particulate PDE4 activity in the prefrontal cortex supports the 5-HT4R-induced increase in information acquisition.

Long-term evaluation of sensorimotor and mnesic behaviour following striatal NMDA-induced unilateral excitotoxic lesion in the mouse.

Excitotoxic lesion of the striatum provides a useful model for evaluating the excitotoxic processes involved in neurological disorders, in particular stroke diseases. The behavioural outcome after such injury is however poorly described. We have therefore investigated the potential behavioural deficits induced by a NMDA-induced excitotoxic unilateral lesion of the lateral part of the striatum, by comparison with a PBS striatal injection (sham procedure), and non-operated mice behaviour. Three groups of male adult Swiss mice were constituted: unilateral NMDA (20 nmol striatal NMDA injection), sham (striatal PBS injection), and control (healthy non-operated mice). From 14 to 29 days post-surgery, sensorimotor and mnesic tests were performed in all groups. After euthanasia, immunohistochemical stainings (NeuN and GFAP) were performed in order to assess the size of the lesion. Straight runway and passive avoidance performances revealed mild deficits related to the excitotoxic NMDA-induced lesion as compared to the sham procedure. Moreover, accelerated rotarod and Morris water maze acquisition performances also revealed deficits related to the surgery, i.e. observed in sham-operated as compared to control mice. NeuN staining revealed no striatal lesion in the sham and non-operated groups in contrast to the NMDA-injected group in which the volume of infarcted striatum was 2.4+/-0.3mm3. GFAP staining revealed a glial reaction in the lesioned striatum of NMDA animals and at the PBS injection site in sham animals. These results suggest that NMDA-induced excitotoxic lesion induces subtle long-term behavioural deficits in mice. Moreover, this study shows the importance of the sham group to investigate the behavioural deficits after excitotoxic lesion models in mice.

Sensorimotor and cognitive deficits after transient middle cerebral artery occlusion in the mouse.

Whereas behavioral impairments after stroke are increasingly studied in the rat, little is known about the long-term functional consequences of focal ischemia in the mouse. To address this issue, Swiss mice underwent transient (60 min) intraluminal occlusion of the middle cerebral artery (MCAo) or sham surgery. Sensorimotor (chimney, accelerating rotarod, pole, corner, adhesive removal and staircase tests) and cognitive (passive avoidance and Morris water maze) performances were regularly assessed during 1 month, after which the final histological lesion was measured. Motor coordination and balance, assessed by the chimney and rotarod tests, were transiently altered by MCAo. Moreover, bradykinesia was evidenced by the pole test. The most striking and long-lasting (1 month) sensorimotor deficits were postural asymmetries on the corner test, bilateral skilled forepaw reaching deficits on the staircase test and a contralateral sensorimotor impairment on the adhesive removal test. MCAo animals showed normal spatial learning abilities on the Morris water maze test, but they displayed learning deficits measured by the passive avoidance test. This latter deficit was significantly correlated with both cortical and striatal damage. Our findings demonstrate the usefulness of three tests that had never been reported in the mouse after ischemia: the adhesive removal, staircase and pole tests, which showed deficits 1 month after ischemia and should therefore constitute meaningful tools in mice for assessing both neuroprotective and regenerative therapies in stroke preclinical studies.

New benzo[h][1,6]naphthyridine and azepino[3,2-c]quinoline derivatives as selective antagonists of 5-HT4 receptors: binding profile and pharmacological characterization.

A series of benzo[h][1,6]naphthyridine and azepino[3,2-c]quinoline derivatives were prepared and evaluated to determine the necessary requirements for high affinity on the 5-HT(4) receptors and high selectivity versus other receptors. The compounds were synthesized by substituting the chlorine atom of benzonaphthyridines and azepinoquinolines with various N-alkyl-4-piperidinylmethanolates. They were evaluated in binding assays with [(3)H]GR 113808 as the 5-HT(4) receptor radioligand. The affinity values (K(i) or inhibition percentages) depended upon the substituent on the aromatic ring on one hand and the substituent on the lateral piperidine chain on the other hand. A chlorine atom produced a marked drop in activity while a N-propyl or N-butyl group gave compounds with nanomolar affinities (1 < K(i) < 10 nM). Among the most potent ligands (3a, 4a, 5a), 4a was selected on the basis of its high affinity and selectivity for pharmacological screening and was evaluated in vivo in specific tests. This compound reveals itself as an antagonist/low partial agonist in the COS-7 cells stably expressing the 5-HT(4(a)) receptor. Derivative 4a also showed in vivo potent analgesic activity in the writhing test at very low doses.