Luis J. Santín

Behavioral phenotype of maLPA1‐null mice: increased anxiety‐like behavior and spatial memory deficits

Lysophosphatidic acid (LPA) has emerged as a new regulatory molecule in the brain. Recently, some studies have shown a role for this molecule and its LPA1 receptor in the regulation of plasticity and neurogenesis in the adult brain. However, no systematic studies have been conducted to investigate whether the LPA1 receptor is involved in behavior. In this study, we studied the phenotype of maLPA1‐null mice, which bear a targeted deletion at the lpa1 locus, in a battery of tests examining neurologic performance, habituation in exploratory behavior in response to low and mild anxiety environments and spatial memory. MaLPA1‐null mutants showed deficits in both olfaction and somesthesis, but not in retinal or auditory functions. Sensorimotor co‐ordination was impaired only in the equilibrium and grasping reflexes. The mice also showed impairments in neuromuscular strength and analgesic response. No additional differences were observed in the rest of the tests used to study sensoriomotor orientation, limb reflexes and co‐ordinated limb use. At behavioral level, maLPA1‐null mice showed an impaired exploration in the open field and increased anxiety‐like response when exposed to the elevated plus maze. Furthermore, the mice exhibit impaired spatial memory retention and reduced use of spatial strategies in the Morris water maze. We propose that the LPA1 receptor may play a major role in both spatial memory and response to anxiety‐like conditions.

Effects of mammillary body lesions on spatial reference and working memory tasks

This work examines the effects of electrolytic mammillary body (MB) lesions on the performance of rats in different spatial memory tasks in the Morris water maze. The first experiment assessed the effect of MB lesion on performance in a spatial reference memory task (place learning with multiple trials). The second experiment examined the effect of a lesion in this nucleus on performance in a spatial working memory task (single-trial place learning). The results show that lesion of the MB impairs the animals performance in spatial working memory tasks but does not impair acquisition in spatial reference memory tasks (place learning, transfer task, reversal task) or in a visual-cued task. However, the deficit in the spatial working memory task does not appear to vary with the delay between acquisition and retention trials (30 s and 5 min). Our results demonstrate a clear role of the mammillary bodies in the processing of spatial information in a working memory task. Lesion of the MB impairs performance in a working memory task but does not affect reference memory processes.

Effects of chronic alcohol consumption on spatial reference and working memory tasks.

The aim of this work was to determine the spatial memory impairments induced by chronic alcohol consumption in rats. The alcoholization process began on the 21st postnatal day and alcohol concentrations were gradually increased to reach a concentration of 20% that was maintained for 4 mon. Behavioral tests were performed in the Morris Water Maze (MWM). The first study assessed the effects of chronic alcohol intake on two reference memory tasks (a place learning with multiple trials and a new place learning carried out in the same experimental context). Alcohol-treated animals presented no overall impairment in their ability to process spatial information. Deficits were restricted to reduced behavioral flexibility in spatial strategies. The second study assessed working memory in two tasks in which information about platform location was only valid for one trial. In the first working memory task, the animals had to perform one trial per day and in the second task they were submitted to four trials per day. At the end of the second experiment, all animals were trained in a visual-cued task. In the second experiment, the most important deficits in alcohol-treated animals occur in spatial working memory tasks, and this impairment was independent of the intertrial interval used. In the second spatial working memory task, performance of the alcohol-treated animals in the earlier trials affected their performance in subsequent trials, suggesting that a process of proactive interference had taken place. The visual-cued task demonstrated that these behavioral impairments were produced without visuoperceptive impairments.

Aggravation of Chronic Stress Effects on Hippocampal Neurogenesis and Spatial Memory in LPA1 Receptor Knockout Mice

Background The lysophosphatidic acid LPA1 receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA1 receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory. Methodology/Principal Findings Male LPA1-null (NULL) and wild-type (WT) mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day). Immunohistochemistry for bromodeoxyuridine and endogenous markers was performed to examine hippocampal cell proliferation, survival, number and maturation of young neurons, hippocampal structure and apoptosis in the hippocampus. Corticosterone levels were measured in another a separate cohort of mice. Finally, the hole-board test assessed spatial reference and working memory. Under control conditions, NULL mice showed reduced cell proliferation, a defective population of young neurons, reduced hippocampal volume and moderate spatial memory deficits. However, the primary result is that chronic stress impaired hippocampal neurogenesis in NULLs more severely than in WT mice in terms of cell proliferation; apoptosis; the number and maturation of young neurons; and both the volume and neuronal density in the granular zone. Only stressed NULLs presented hypocortisolemia. Moreover, a dramatic deficit in spatial reference memory consolidation was observed in chronically stressed NULL mice, which was in contrast to the minor effect observed in stressed WT mice. Conclusions/Significance These results reveal that the absence of the LPA1 receptor aggravates the chronic stress-induced impairment to hippocampal neurogenesis and its dependent functions. Thus, modulation of the LPA1 receptor pathway may be of interest with respect to the treatment of stress-induced hippocampal pathology.

When is adult hippocampal neurogenesis necessary for learning? Evidence from animal research

Abstract The hippocampus is a key brain structure involved in the short- and long-term processing of declarative memory. Since adult hippocampal neurogenesis was first found, numerous studies have tried to establish the contribution of newborn neurons to hippocampus-dependent cognitive functions. However, this large amount of research has generated contradictory results. In this paper, we review the body of evidence investigating the relationship between hippocampal neurogenesis and learning to conclude the functional role of adult-born hippocampal neurons. First, factors that could explain discrepancies among experiments are taken into account. Then, in addition to methodological differences, we emphasize the importance of the age of the newborn neurons studied, as to how their maturation influences both their properties and potential functionality. Next, we discuss which declarative memory components could require involvement of adult hippocampal neurogenesis, taking into consideration the representational demands of the task, its difficulty and the level of performance reached by the subject. Finally, other factors that could modulate neurogenesis and memory, such as stress levels or previous experience of the animal, should also be taken into consideration in interpreting experiments focused on neurogenesis. In conclusion, our analysis of published studies suggests that new adult-born neurons, under certain circumstances, have a crucial and irreplaceable role in hippocampal learning.

c-Fos expression in supramammillary and medial mammillary nuclei following spatial reference and working memory tasks.

To investigate brain substrates of spatial memory, neuronal expression of c-Fos protein was studied. Two groups of rats were trained in two spatial memory tasks in the Morris water maze, where the rats have to apply a reference memory rule or a working memory rule. In addition to the experimental groups, two control groups were used to study c-fos activation not specific to the memory processes studied. After immunohistochemical procedures, the number of c-Fos positive neuronal nuclei was quantified in the mammillary body (MB) region (medial mammillary nucleus [MMn] and supramammillary nucleus [SuM]). The results have shown that some MMn neurons expressed c-Fos nuclear immunoreactivity related to spatial working memory but not to spatial reference memory. The increased number of c-Fos immunoreactive neuronal nuclei in the SuM was related to spatial training but not to either working or reference memory demands of the tasks.

Improvement of spatial memory by (R)-α-methylhistamine, a histamine H3-receptor agonist, on the Morris water-maze in rat

This work aims to clarify the role of histamine in learning and memory. This is done by studying the effect of administration of the histamine precursor, L-histidine (His), and the agonist of the H(3) receptor (R)-alpha-methylhistamine (RAMH), on acquisition and retention of spatial reference memory in rats. Treatment with RAMH (10 mg/kg i.p.) facilitates recovery of spatial memory. In contrast, administration of His (500 mg/kg i.p.) does not affect the performance of this task. Moreover, pharmacological modulation of the cerebral histaminergic system does not impair the animals behavioral flexibility, i.e. their ability to adapt to a new learning task in the same stimular context. Improved retention of spatial memory after a reduction in cerebral histamine confirms the modulating role of this neurotransmitter in memory processes.

Attenuation of cocaine-induced conditioned locomotion is associated with altered expression of hippocampal glutamate receptors in mice lacking LPA1 receptors

RationaleLysophosphatidic acid is a phospholipid mediator that modulates neurodevelopment and neurogenesis in the hippocampus through its actions on LPA1 receptors. Emerging evidences support LPA1 as a mediator of learning and emotional behaviour. There are no studies addressing its role on behaviours associated to drug abuse.ObjectivesWe examined whether genetic deletion of LPA1 receptor in maLPA1-null mice affected either cocaine-induced conditioned locomotion (CL) or behavioural sensitization (BS) induced by repeated cocaine exposure. We also analysed whether cocaine induced changes in the expression of functional markers of both dopamine- and glutamate-related genes in the striatum and the dorsal hippocampus.MethodsWe monitored cocaine-induced CL and BS in both genotypes of mice. Striatal dopamine and hippocampal glutamate-related genes were measured by real-time quantitative PCR, Western blot, and immunohistochemistry.ResultsmaLPA1-null mice exhibit an attenuated CL response after cocaine conditioning but a normal BS after repeated cocaine exposure. These behavioural changes were associated to alterations on the expression of metabotropic mGLUR3 glutamate receptors and on the actions of cocaine on the GLUR1 subunit of AMPA glutamate receptors in the hippocampus of maLPA1 animals. Striatal dopaminergic markers (tyrosine hydroxylase, dopamine D1 receptor, and dopamine transporter DAT), were similar in both genotypes and were equally affected by cocaine exposure.ConclusionThe present results indicate that the lack of LPA1 receptor affect cocaine-induced conditioned locomotion but not behavioural sensitization. The findings suggest that LPA1 receptor may be necessary for a normal associative contextual learning associated to cocaine, probably through the modulation of hippocampal glutamatergic circuits.

Hippocampal c-Fos activation in normal and LPA1-null mice after two object recognition tasks with different memory demands

Normal and LPA₁-null mice, that have well reported hippocampal deficits, were assessed in an episodic-like what-when-where memory task or in a comparable task designed to test memory for familiar objects and locations by discriminating them from novels. Both genotypes performed the novelty recognition task but failed to learn the what-when-where task. However, normal mice showed what-when memory that was impaired in nulls. Each task elicited a different pattern of c-Fos expression. In normal mice, the what-when-where task induced more hippocampal c-Fos activation in the CA1 area than the novelty-based task, correlating with the what-when memory. LPA₁-null mice displayed a basal c-Fos hyperactivity in the hippocampus and in the medial prefrontal cortex, which was regulated differently by the two behavioural tasks employed. Both tasks were matched in exploratory behaviour and c-Fos activation in stress-related brain areas for both genotypes. This study shows that the what-when-where memory task differs from a comparable novelty-based task in both the learning demands and the neuronal correlates. Moreover, results also stress the role of the LPA₁ receptor in hippocampal functioning.

A Role for Galanin N-Terminal Fragment (1–15) in Anxiety- and Depression-Related Behaviors in Rats

Background: Galanin (GAL) plays a role in mood regulation. In this study we analyzed the action of the active N-terminal fragment [GAL(1–15)] in anxiety- and depression-related behavioral tests in rats. Methods: The effect of GAL(1–15) was analyzed in the forced swimming test, tail suspension test, open field test, and light/dark test. The proximity of GAL1 and GAL2 receptors was examined with the proximity ligation assay (PLA). We tested the GAL receptors involved in GAL(1–15) effects with the GAL2 receptor antagonist M871 and with an in vivo model of siRNA GAL2 receptor knockdown or siRNA GAL1 receptor knockdown rats. The effects of GAL(1–15) were also studied in the cell line RN33B. Results: GAL(1–15) induced strong depression-like and anxiogenic-like effects in all the tests. These effects were stronger than the ones induced by GAL. The involvement of the GAL2 receptor was demonstrated with M871 and with the siRNA GAL2 receptor knockdown rats. The PLA indicated the possible existence of GAL1 and GAL2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe nucleus. In the siRNA GAL1 receptor knockdown rats the behavioral actions of GAL(1–15) disappeared, and in the siRNA GAL2 receptor knockdown rats the reductions of the behavioral actions of GAL(1–15) was linked to a disappearance of PLA. In the cell line RN33B, GAL(1–15) decreased 5-HT immunoreactivity more strongly than GAL. Conclusions: Our results indicate that GAL(1–15) exerts strong depression-related and anxiogenic-like effects and may give the basis for the development of drugs targeting GAL1 and GAL2 heteroreceptor complexes in the raphe-limbic system for the treatment of depression and anxiety.