Estela Castilla-Ortega

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.

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.

Pharmacological blockade of either cannabinoid CB1 or CB2 receptors prevents both cocaine-induced conditioned locomotion and cocaine-induced reduction of cell proliferation in the hippocampus of adult male rat.

Addiction to major drugs of abuse, such as cocaine, has recently been linked to alterations in adult neurogenesis in the hippocampus. The endogenous cannabinoid system modulates this proliferative response as demonstrated by the finding that pharmacological activation/blockade of cannabinoid CB1 and CB2 receptors not only modulates neurogenesis but also modulates cell death in the brain. In the present study, we evaluated whether the endogenous cannabinoid system affects cocaine-induced alterations in cell proliferation. To this end, we examined whether pharmacological blockade of either CB1 (Rimonabant, 3 mg/kg) or CB2 receptors (AM630, 3 mg/kg) would affect cell proliferation [the cells were labeled with 5-bromo-2′-deoxyuridine (BrdU)] in the subventricular zone (SVZ) of the lateral ventricle and the dentate subgranular zone (SGZ). Additionally, we measured cell apoptosis (as monitored by the expression of cleaved caspase-3) and glial activation [by analyzing the expression of glial fibrillary acidic protein (GFAP) and Iba-1] in the striatum and hippocampus during acute and repeated (4 days) cocaine administration (20 mg/kg). The results showed that acute cocaine exposure decreased the number of BrdU-immunoreactive (ir) cells in the SVZ and SGZ. In contrast, repeated cocaine exposure reduced the number of BrdU-ir cells only in the SVZ. Both acute and repeated cocaine exposure increased the number of cleaved caspase-3-, GFAP- and Iba1-ir cells in the hippocampus, and this effect was counteracted by AM630 or Rimonabant, which increased the number of BrdU-, GFAP-, and Iba1-ir cells in the hippocampus. These results indicate that the changes in neurogenic, apoptotic and gliotic processes that were produced by repeated cocaine administration were normalized by pharmacological blockade of CB1 and CB2. The restorative effects of cannabinoid receptor blockade on hippocampal cell proliferation were associated with the prevention of the induction of conditioned locomotion but not with the prevention of cocaine-induced sensitization.

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.

Pharmacological reduction of adult hippocampal neurogenesis modifies functional brain circuits in mice exposed to a cocaine conditioned place preference paradigm

We investigated the role of adult hippocampal neurogenesis in cocaine‐induced conditioned place preference (CPP) behaviour and the functional brain circuitry involved. Adult hippocampal neurogenesis was pharmacologically reduced with temozolomide (TMZ), and mice were tested for cocaine‐induced CPP to study c‐Fos expression in the hippocampus and in extrahippocampal addiction‐related areas. Correlational and multivariate analysis revealed that, under normal conditions, the hippocampus showed widespread functional connectivity with other brain areas and strongly contributed to the functional brain module associated with CPP expression. However, the neurogenesis‐reduced mice showed normal CPP acquisition but engaged an alternate brain circuit where the functional connectivity of the dentate gyrus was notably reduced and other areas (the medial prefrontal cortex, accumbens and paraventricular hypothalamic nucleus) were recruited instead of the hippocampus. A second experiment unveiled that mice acquiring the cocaine‐induced CPP under neurogenesis‐reduced conditions were delayed in extinguishing their drug‐seeking behaviour. But if the inhibited neurons were generated after CPP acquisition, extinction was not affected but an enhanced long‐term CPP retention was found, suggesting that some roles of the adult‐born neurons may differ depending on whether they are generated before or after drug–contextual associations are established. Importantly, cocaine‐induced reinstatement of CPP behaviour was increased in the TMZ mice, regardless of the time of neurogenesis inhibition. The results show that adult hippocampal neurogenesis sculpts the addiction‐related functional brain circuits, and reduction of the adult‐born hippocampal neurons increases cocaine seeking in the CPP model.

Effects of medial prefrontal cortex lesions on anxiety-like behaviour in restrained and non-restrained rats.

The medial prefrontal cortex has been associated with fear, anxiety and stress regulation, and has recently been suggested to play a crucial role in the development of behavioural changes in response to stress. In this study, we evaluated medial prefrontal cortex (mPFC) involvement in both anxiety-like behaviour and increased anxiety-like responses induced by uncontrollable restraint. Rats with mPFC electrolytic lesions (n=7) and sham-lesioned (n=8) were tested in the elevated T-maze (ETM). Restrained rats with mPFC lesions (n=8) and sham-lesioned rats (n=6) were tested in the elevated T-maze 24h after restraint. Both two-trial passive avoidance and one-trial escape behaviours were assessed. The results revealed that mPFC lesions impair passive avoidance, but not escape behaviour. In addition, decreased anxiety-like behaviour in both passive avoidance and escape behaviours were observed in restrained rats with mPFC lesions. Our results suggest that mPFC is important in mediating both anxiety-like behaviour expression and long-term anxiogenic-like effects induced by acute restraint.

Fear extinction and acute stress reactivity reveal a role of LPA(1) receptor in regulating emotional-like behaviors.

LPA1 receptor is one of the six characterized G protein-coupled receptors (LPA1–6) through which lysophosphatidic acid acts as an intercellular signaling molecule. It has been proposed that this receptor has a role in controlling anxiety-like behaviors and in the detrimental consequences of stress. Here, we sought to establish the involvement of the LPA1 receptor in emotional regulation. To this end, we examined fear extinction in LPA1-null mice, wild-type and LPA1 antagonist-treated animals. In LPA1-null mice we also characterized the morphology and GABAergic properties of the amygdala and the medial prefrontal cortex. Furthermore, the expression of c-Fos protein in the amygdala and the medial prefrontal cortex, and the corticosterone response following acute stress were examined in both genotypes. Our data indicated that the absence of the LPA1 receptor significantly inhibited fear extinction. Treatment of wild-type mice with the LPA1 antagonist Ki16425 mimicked the behavioral phenotype of LPA1-null mice, revealing that the LPA1 receptor was involved in extinction. Immunohistochemistry studies revealed a reduction in the number of neurons, GABA+ cells, calcium-binding proteins and the volume of the amygdala in LPA1-null mice. Following acute stress, LPA1-null mice showed increased corticosterone and c-Fos expression in the amygdala. In conclusion, LPA1 receptor is involved in emotional behaviors and in the anatomical integrity of the corticolimbic circuit, the deregulation of which may be a susceptibility factor for anxiety disorders and a potential therapeutic target for the treatment of these diseases.

Chronic immobilization in the malpar1 knockout mice increases oxidative stress in the hippocampus.

ABSTRACT The lysophosphatidic acid LPA1 receptor has recently been involved in the adaptation of the hippocampus to chronic stress. The absence of LPA1 receptor aggravates the chronic stress-induced impairment of both hippocampal neurogenesis and apoptosis that were accompanied with hippocampus-dependent memory deficits. Apoptotic death and neurogenesis in the hippocampus are regulated by oxidative stress. In the present work, we studied the involvement of LPA1 receptor signaling pathway in the regulation of the hippocampal redox after chronic stress. To this end, we used malpar1 knockout (KO) and wild-type mice assigned to either chronic stress (21 days of restraint, 3 h/day) or control conditions. Lipid peroxidation, the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX), as well as mitochondrial function stimulation, monitored through the activity of cytochrome c oxidase (COX), were studied in the hippocampus. Our results showed that chronic immobilization stress enhanced lipid peroxidation as well as the activity of the antioxidant enzymes studied (CAT, SOD, and GPX). This effect was only observed in absence of LPA1 receptor. Furthermore, only malpar1 KO mice submitted to chronic stress exhibited a severe downregulation of the COX activity, suggesting the presence of mitochondrial damage. Altogether, these results suggest that malpar1 KO mice display enhanced oxidative stress in the hippocampus after chronic stress. This may be involved in the hippocampal abnormalities observed in this genotype after chronic immobilization, including memory, neurogenesis, and apoptosis.

Sex differences in psychiatric comorbidity and plasma biomarkers for cocaine addiction in abstinent cocaine-addicted subjects in outpatient settings

There are sex differences in the progression of drug addiction, relapse, and response to therapies. Because biological factors participate in these differences, they should be considered when using biomarkers for addiction. In the current study, we evaluated the sex differences in psychiatric comorbidity and the concentrations of plasma mediators that have been reported to be affected by cocaine. Fifty-five abstinent cocaine-addicted subjects diagnosed with lifetime cocaine use disorders (40 men and 15 women) and 73 healthy controls (48 men and 25 women) were clinically assessed with the diagnostic interview “Psychiatric Research Interview for Substance and Mental Disorders.” Plasma concentrations of chemokines, cytokines, N-acyl-ethanolamines, and 2-acyl-glycerols were analyzed according to history of cocaine addiction and sex, controlling for covariates age and body mass index (BMI). Relationships between these concentrations and variables related to cocaine addiction were also analyzed in addicted subjects. The results showed that the concentrations of chemokine (C-C motif) ligand 2/monocyte chemotactic protein-1 (CCL2/MCP-1) and chemokine (C-X-C motif) ligand 12/stromal cell-derived factor-1 (CXCL12/SDF-1) were only affected by history of cocaine addiction. The plasma concentrations of interleukin 1-beta (IL-1β), IL-6, IL-10, and tumor necrosis factor-alpha (TNFα) were affected by history of cocaine addiction and sex. In fact, whereas cytokine concentrations were higher in control women relative to men, these concentrations were reduced in cocaine-addicted women without changes in addicted men. Regarding fatty acid derivatives, history of cocaine addiction had a main effect on the concentration of each acyl derivative, whereas N-acyl-ethanolamines were increased overall in the cocaine group, 2-acyl-glycerols were decreased. Interestingly, N-palmitoleoyl-ethanolamine (POEA) was only increased in cocaine-addicted women. The covariate BMI had a significant effect on POEA and N-arachidonoyl-ethanolamine concentrations. Regarding psychiatric comorbidity in the cocaine group, women had lower incidence rates of comorbid substance use disorders than did men. For example, alcohol use disorders were found in 80% of men and 40% of women. In contrast, the addicted women had increased prevalences of comorbid psychiatric disorders (i.e., mood, anxiety, and psychosis disorders). Additionally, cocaine-addicted subjects showed a relationship between the concentrations of N-stearoyl-ethanolamine and 2-linoleoyl-glycerol and diagnosis of psychiatric comorbidity. These results demonstrate the existence of a sex influence on plasma biomarkers for cocaine addiction and on the presence of comorbid psychopathologies for clinical purposes.