Gating of Sema3E/PlexinD1 signaling by neuropilin-1 switches axonal repulsion to attraction during brain development.

The establishment of functional neural circuits requires the guidance of axons in response to the actions of secreted and cell-surface molecules such as the semaphorins. Semaphorin 3E and its receptor PlexinD1 are expressed in the brain, but their functions are unknown. Here, we show that Sema3E/PlexinD1 signaling plays an important role in initial development of descending axon tracts in the forebrain. Early errors in axonal projections are reflected in behavioral deficits in Sema3E null mutant mice. Two distinct signaling mechanisms can be distinguished downstream of Sema3E. On corticofugal and striatonigral neurons expressing PlexinD1 but not Neuropilin-1, Sema3E acts as a repellent. In contrast, on subiculo-mammillary neurons coexpressing PlexinD1 and Neuropilin-1, Sema3E acts as an attractant. The extracellular domain of Neuropilin-1 is sufficient to convert repulsive signaling by PlexinD1 to attraction. Our data therefore reveal a "gating" function of neuropilins in semaphorin-plexin signaling during the assembly of forebrain neuronal circuits.

Spatial memory in the Morris water maze and activation of cyclic AMP response element-binding (CREB) protein within the mouse hippocampus.

We investigated the spatio-temporal dynamics of learning-induced cAMP response element-binding protein activation/phosphorylation (pCREB) in mice trained in a spatial reference memory task in the water maze. Using immunohistochemistry, we examined pCREB immunoreactivity (pCREB-ir) in hippocampal CA1 and CA3 and related brain structures. During the course of spatial learning over Days 1-9, pCREB-ir progressively increased in hippocampal neurons whereas its level in the dorsal striatum decreased. No significant changes were observed in the prelimbic cortex and lateral amygdala. Mice killed at various time points after the last training session demonstrated two waves of pCREB-ir in CA1 and an early transient CREB phosphorylation in area CA3, lateral amygdala, and prelimbic cortex. We show that CREB phosphorylation and downstream gene Zif268 activation remained sustained in CA1 and CA3 for at least 24 h after extended training (Days 8-9) but not during early training (Day 3). The present results indicate that the strong CA1 CREB phosphorylation observed immediately after training was not related strictly to learning or to memory. In contrast, at 15 min after training, the changes in CA1 CREB phosphorylation state were specifically related to individual learning capability. We suggest that hippocampal-learning specificity of CREB is reflected best by duration, rather than magnitude, of CREB phosphorylation.

Presynaptic serotonergic modulation of 5-HT and acetylcholine release in the hippocampus and the cortex of 5-HT1B-receptor knockout mice.

Lesioning of serotonergic afferents increases hippocampal ACh release and attenuates memory deficits produced by cholinergic lesions. Improved memory performance described in 5-HT1B-knockout (KO) mice might thus be due to a weaker 5-HT1B-mediated inhibitory influence of 5-HT on hippocampal ACh release. The selective delay-dependent impairment of working memory observed in these KO mice suggests, however, that cortical regions also participate in task performance, possibly via indirect influences of 5-HT on ACh release. To provide neuropharmacological support for these hypotheses we measured evoked ACh and 5-HT release in hippocampal and cortical slices of wild-type (WT) and 5-HT1B KO mice. Superfused slices (preincubated with [3H]choline or [3H]5-HT) were electrically stimulated in the absence or presence of 5-HT1B receptor ligands. In hippocampus and cortex, 5-HT1B agonists decreased and antagonists increased 5-HT release in WT, but not in 5-HT1B KO mice. In 5-HT1B KO mice, 5-HT release was enhanced in both structures, while ACh release (in nCi) was reduced. ACh release was inhibited by 5-HT1B agonists in hippocampal (not cortical) slices of WT but not of 5-HT1B KO mice. Our data (i) confirm the absence of autoinhibition of 5-HT release in 5-HT1B-KO mice, (ii) demonstrate a reduced release of ACh, and the absence of 5-HT1B-receptor-mediated inhibition of ACh release, in the hippocampus and cortex of 5-HT1B-KO mice, and (iii) are compatible with an indirect role of cortical ACh in the working memory impairment observed in these KO mice.

Age-dependent effects of serotonin-1A receptor gene deletion in spatial learning abilities in mice.

The serotonin (5-hydroxytryptamine, 5-HT) receptor 1A is involved in many physiological functions, including the regulation of learning and memory by acting either as an autoreceptor located on 5-HT neurons (raphe nuclei) or as a heteroreceptor on non-5-HT neurons, mainly in the hippocampal formation. To investigate whether the effects of 5-HT via 5-HT1A receptors on learning are age-sensitive, we evaluated the performance of young-adult (3 months old) and aged (22 months old) 5-HT1A knockout (KO) mice and their homologous wild types (WT) in the hippocampal-dependent spatial reference memory version of the Morris water maze. We demonstrated that young-adult 5-HT1AKO mice exhibit an impairment in learning and retention of the spatial task, as compared to WT mice, without showing any sign of change in their sensori-motor and locomotor abilities or motivation. This genotype effect does not persist during aging. In fact, aged 5-HT1AKO mice seem to be slightly facilitated during the early stages of learning. These results are consistent with a possible prevalence of 5-HT1A raphe functions in learning and memory abilities of young-adult animals, since the effects of the mutation on mice performance (impairment) are opposite to those found after intra-raphe injection of 5-HT1A agonists (facilitation), and with data showing increased activity of 5-HT neurons in 5-HT1AKO mice. The reduced effect of the mutation in aged animals possibly reflects the lower efficacy of autoreceptors due to aging and/or a prevalence of hippocampal heteroreceptors.

Rewarding effects elicited by cocaine microinjections into the ventral tegmental area of C57BL/6 mice: involvement of dopamine D1 and serotonin1B receptors.

RATIONALE: The role of ventral tegmental area (VTA) in mediating the rewarding effects of cocaine has not been extensively studied. OBJECTIVES: We used the intracranial self-administration (ICSA) procedure to assess the involvement of the VTA in the rewarding effects of cocaine, and the effect of dopamine (DA) D(1)- and serotonin (5-HT)(1B)-receptor antagonists on ICSA of cocaine. METHODS: Adult male C57BL/6 mice were stereotaxically implanted, unilaterally, with a guide cannula either 1.5 or 2.3 mm above the VTA. After 1 week, mice were trained to discriminate between the two arms of a Y-maze over seven daily sessions, one arm being reinforced by intracranial cocaine microinjections. Starting from session 8, the D(1) and 5-HT(1B)-receptor antagonists were injected IP pre-test each day over five consecutive sessions. RESULTS: Mice injected into the VTA rapidly exhibited a preference for the cocaine-reinforced arm, whatever the dose of cocaine available (30 pmol or 150 pmol per injection), reaching optimum ICSA performance within 5 days. In contrast, mice injected 0.8 mm above the VTA did not discriminate between the arms of the maze and performed at random, except for one subject. Once the ICSA response was acquired, systemic pre-injections of either the D(1) (SCH23390; 25 microg/kg IP) or 5-HT(1B) (GR127935; 0.5 mg/kg IP) antagonist disrupted this behavior. Replacement of each antagonist by vehicle led to the reinstatement of intra-VTA cocaine self-administration. CONCLUSIONS: The results of the present study suggest that VTA neurons play a critical role in mediating the rewarding effects of acute cocaine and that both D(1) and 5-HT(1B) receptors modulate these effects.

Interaction between the nature of the information and the cognitive requirement of the task in problem solving in mice.

The Morris water maze and the radial-arm maze are two of the most frequently employed behavioral tasks used to assess spatial memory in rodents. In this study, we describe two new behavioral tasks in a radial-arm water maze enabling to combine the advantages of the Morris water maze and the radial-arm maze. In both tasks, spatial and nonspatial learning was assessed and the only task parameter that varied was the nature of the information available which was either spatial (various distal extra-maze cues) or nonspatial (visual intra-maze patterns). In experiment 1, 129T2/Sv mice were able to learn three successive pairwise discriminations [(1) A+/B-, (2) B+/C-, (3) C+/A-] with the same efficiency in both modalities (i.e. spatial and nonspatial modalities). Probe-trials at the end of each of these discriminations revealed particular features of this transverse-patterning-like procedure. In experiment 2, another group of 129T2/Sv mice was submitted to a delayed matching-to-sample working memory task. Mice were able to learn the task and were then able to show resistance to temporal interference as long as 60 min in the spatial modality but they failed to acquire the task in the nonspatial modality. The fact that the nonspatial information was exactly the same in both experiments highlights the existence of an interaction between the cognitive requirements of the task and the nature of the information.

Differential learning abilities of 129T2/Sv and C57BL/6J mice as assessed in three water maze protocols.

Knockout mice are generated by using ES cells from 129 mouse strains and are frequently backcrossed with other strains, like C57BL/6. It is important to characterise the physiological and, in particular, the behavioural profile of each strain in order to correctly analyse the functional contribution of a single gene mutation on the 'cognitive' phenotype. The present study compared 129T2/Sv (129) and C57BL/6J (C57) mice in three different spatial learning protocols in the water maze, using a hidden platform. In the 'standard' reference memory protocol, 129 and C57 attained an equivalent level of performance as assessed by accuracy in reaching the platform (path length), despite a faster swim speed exhibited by C57 mice. In a stepwise learning task, C57 mice showed poorer performances over all stages of learning. However they performed better than 129 in a massed learning protocol which taxes short-term memory, and in which they exhibited lower levels of perseveration. The results emphasize the importance of using various tasks differing in cognitive demand, but using the same experimental environment and motivation, in order to 1) evaluate strain- or mutation-dependent learning abilities, and 2) dissociate the roles played by cognitive and non-cognitive factors in the behavioural requirements of the tasks.

Protective effect of 5-HT1B receptor gene deletion on the age-related decline in spatial learning abilities in mice.

We previously observed that 5 months old serotonin 1B receptor knockout (5-HT1BKO) mice exhibited a facilitation of learning in a long-term spatial memory task in a water maze. In this study, we attempted to assess whether this effect might persist during aging. We compared the performances of young-adult (3 months old) and aged (22 months old) 5-HT1BKO and wild type (WT) mice in the same task. Young-adult and aged KO mice exhibited facilitated acquisition of the reference memory task as compared to their respective WT controls. Generally, the performance of aged KO was similar to that of young-adult WT on the parameters defining performance and motor (swim speed) aspects of the task. During probe trials, all mice presented a spatial selectivity, which was, however, less pronounced in aged than in young-adult WT. No such age-related effect was observed in KO mice. In a massed spatial learning task, aged KO and WT mice globally exhibited the same level of performance. Nevertheless, young-adult and aged KO mice were superior to their WT controls as concerns the working memory component of the task. The data suggest that 5-HT1BKO mice are more resistant than WT to age-related memory decline as concerns both reference/long-term and working/short-term spatial memory.

Hyperfunction of muscarinic receptor maintains long-term memory in 5-HT4 receptor knock-out mice.

Patients suffering from dementia of Alzheimer's type express less serotonin 4 receptors (5-HTR(4)), but whether an absence of these receptors modifies learning and memory is unexplored. In the spatial version of the Morris water maze, we show that 5-HTR(4) knock-out (KO) and wild-type (WT) mice performed similarly for spatial learning, short- and long-term retention. Since 5-HTR(4) control mnesic abilities, we tested whether cholinergic system had circumvented the absence of 5-HTR(4). Inactivating muscarinic receptor with scopolamine, at an ineffective dose (0.8 mg/kg) to alter memory in WT mice, decreased long-term but not short-term memory of 5-HTR(4) KO mice. Other changes included decreases in the activity of choline acetyltransferase (ChAT), the required enzyme for acetylcholine synthesis, in the septum and the dorsal hippocampus in 5-HTR(4) KO under baseline conditions. Training- and scopolamine-induced increase and decrease, respectively in ChAT activity in the septum in WT mice were not detected in the 5-HTR(4) KO animals. Findings suggest that adaptive changes in cholinergic systems may circumvent the absence of 5-HTR(4) to maintain long-term memory under baseline conditions. In contrast, despite adaptive mechanisms, the absence of 5-HTR(4) aggravates scopolamine-induced memory impairments. The mechanisms whereby 5-HTR(4) mediate a tonic influence on ChAT activity and muscarinic receptors remain to be determined.

Alteration of CREB phosphorylation and spatial memory deficits in aged 129T2/Sv mice.

Phosphorylation of cAMP-response element binding protein (CREB) is required for hippocampus-dependent long-term memory formation. The present study was designed to determine whether spatial memory deficits in aged mice were associated with alteration of hippocampal CREB phosphorylation. We examined the temporal pattern of CREB activation in 5-6 and 23-24-month-old 129T2/Sv mice trained on a spatial reference memory task in the water maze. Phosphorylated CREB (pCREB), total CREB (t-CREB) and c-Fos immunoreactivity (ir) were measured at four time points after the end of training. In young mice, pCREB-ir was significantly increased 15 and 60 min after training in the CA1 region and dentate gyrus. In aged mice sacrificed 15 min after training, pCREB-ir in these structures was reduced whereas t-CREB-ir remained unchanged compared to respective young-adults. An age-related reduction of c-Fos-ir also occurred selectively in hippocampal CA1 region. Since reduced pCREB-ir in CA1 from the 15 min-aged group strongly correlated with individual learning performance, we suggest that altered CREB phosphorylation in CA1 may account for spatial memory impairments during normal aging.

Impairment of spatial learning and memory in ELKL Motif Kinase1 (EMK1/MARK2) knockout mice.

The hyperphosphorylation of tau protein is one of the hallmarks of Alzheimer's disease (AD) and of the associated cognitive decline. EMK1 (MARK2) is a serine/threonine kinase which phosphorylates tau and MAP2. An involvement of this kinase in memory functions is not established. We used a behavioral approach to study the phenotype of EMK1-null mice (EMK1-KO) as a possible model of MAP2/tau altered phophorylation. Compared to wild type mice, EMK1-KO mice did not differ in non-cognitive aspects of behavior, such as locomotion in activity cages, or anxiety in the elevated plus maze. However, they exhibited lower performance in the first stage of acquisition of a hippocampal-dependent spatial learning, as assessed in a radial water maze, although, they acquired the task with repeated training. They were again found to be impaired on re-learning a new platform position. In addition, they exhibited poor long-term retention performance. These data underline the importance on both early memory processes and long-term retrieval, of the dynamic instability of microtubules generated by the phosphorylation of MAPs.

Spatial learning in the 5-HT1B receptor knockout mouse: selective facilitation/impairment depending on the cognitive demand.

Age-related memory decline is associated with a combined dysfunction of the cholinergic and serotonergic systems in the hippocampus and frontal cortex, in particular. The 5-HT1B receptor occupies strategic cellular and subcellular locations in these structures, where it plays a role in the modulation of ACh release. In an attempt to characterize the contribution of this receptor to memory functions, 5-HT1B receptor knockout (KO) mice were submitted to various behavioral paradigms carried out in the same experimental context (water maze), which were aimed at exposing mice to various levels of memory demand. 5-HT1BKO mice exhibited a facilitation in the acquisition of a hippocampal-dependent spatial reference memory task in the Morris water maze. This facilitation was selective of task difficulty, showing thus that the genetic inactivation of the 5-HT1B receptor is associated with facilitation when the complexity of the task is increased, and reveals a protective effect on age-related hippocampal-dependent memory decline. Young-adult and aged KO and wild-type (WT) mice were equally able to learn a delayed spatial matching-to-sample working memory task in a radial-arm water maze with short (0 or 5 min) delays. However, 5-HT1BKO mice, only, exhibited a selective memory impairment at intermediate and long (15, 30, and 60 min) delays. Treatment by scopolamine induced the same pattern of performance in wild type as did the mutation for short (5 min, no impairment) and long (60 min, impairment) delays. Taken together, these studies revealed a beneficial effect of the mutation on the acquisition of a spatial reference memory task, but a deleterious effect on a working memory task for long delays. This 5-HT1BKO mouse story highlights the problem of the potential existence of "global memory enhancers."

Delay-dependent working memory impairment in young-adult and aged 5-HT1BKO mice as assessed in a radial-arm water maze.

Serotonin (5-HT) plays a modulatory role in mnemonic functions, especially by interacting with the cholinergic system. The 5-HT1B receptor is a key target of this interaction. The 5-HT1B receptor knockout mice were found previously to exhibit a facilitation in hippocampal-dependent spatial reference memory learning. In the present study, we submitted mice to a delayed spatial working memory task, allowing the introduction of various delays between an exposure trial and a test trial. The 5-HT1BKO and wild-type mice learned the task in a radial-arm water maze (returning to the most recent presented arm containing the escape platform), and exhibited a high level of performance at delays of 0 and 5 min. However, at the delay of 60 min, only 5-HT1BKO mice exhibited an impairment. At a delay of 90 min, all mice were impaired. Treatment by scopolamine (0.8 mg/kg) induced the same pattern of performance in wild type as did the mutation for short (5 min, no impairment) and long (60 min, impairment) delays. The 22-month-old wild-type and knockout mice exhibited an impairment at short delays (5 and 15 min). The effect of the mutation affected both young-adult and aged mice at delays of 15, 30, and 60 min. Neurobiological data show that stimulation of the 5-HT1B receptor inhibits the release of acetylcholine in the hippocampus, but stimulates this in the frontal cortex. This dual function might, at least in part, explain the opposite effect of the mutation on reference memory (facilitation) and delay-dependent working memory (impairment). These results support the idea that cholinergic-serotonergic interactions play an important role in memory processes.