BrdU administration and behavioural analysis protocols.

<p>Analyses of new hippocampal cell proliferation (A) survival (B) and behaviour on the hole-board test (C) were carried out in different groups of mice from both genotypes submitted either to standard housing (control) or 21 days of chronic stress. (A) To assess cell proliferation, BrdU injections (75 mg/kg) were given on the final 4 days of stressor exposure in the chronically stressed groups. All animals were sacrificed by perfusion the day following the final BrdU dose. Brain tissue from these animals was also processed for PCNA, DCX and GFAP immunolabeling and apoptosis. (B) For cell survival assessments, BrdU was administered on the 4 days prior to stressor exposure in the chronically stressed groups, and all animals were sacrificed by perfusion 22 days following the final BrdU injection. These animals were also assessed for NeuN staining. (C) Behavioural analyses were conducted using the hole-board test over 2 days of habituation and 4 days of spatial learning. All holes were baited during habituation, but only 4 were baited during the spatial learning phase.</p

Neuronal and glial phenotype of the newly born hippocampal cells.

<p>Mean values ± SEM. The absence of LPA₁ receptor did not affected the percentage of BrdU labelled cells (aged 1 to 4 days) that had differentiated into immature neurons (A, BrdU co-labelling with DCX) or in astrocytes (B, BrdU co-labelling with a GFAP+ cell with stellar morphology), although chronic stress reduced the glial fate. (- %): reduction from the control group. Scale bar in A (also valid for B): 10 µm. Post-hoc LSD tests: (*<i>p</i><.05): significant difference between the stressed group vs. its control.</p

Cell proliferation and survival in the dentate gyrus.

<p>Mean values ± SEM. (A, B) MaLPA₁-null mice showed defective cell proliferation as assessed by BrdU (A) and PCNA (B) immunolabeling. Notably, the number of proliferating cells was reduced by chronic stress only in the mutant genotype. (C) Chronic stress reduced the survival of new cells labelled with BrdU prior to stress treatment, but cell survival was similar between genotypes when data were corrected for their distinct proliferation rates. (- %): reduction from the control group. Scale bar in A (also valid for B and C): 100 µm; 20 µm. Arrows indicate cells positive for BrdU (A) and PCNA (B). Post-hoc LSD tests: (*<i>p</i><.05; **<i>p</i><.001): significant difference between the stressed group vs. its control; (#<i>p</i><.05; ##<i>p</i><.001): significant difference between the control NULL vs. the control WT mice.</p

Hippocampal structure in control and stressed WT and maLPA₁-null mice in NeuN-stained sections.

<p>Mean values ± SEM. MaLPA₁-null mice showed reduced hippocampal volume in the oriens, pyramidal and radiatum layers in the CA1 and CA3 areas; no structural alterations were observed in the DG. Chronic stress reduced the volume of the pyramidal layer in both genotypes; however, reduced DG granular layer volume was observed only in the stressed NULL genotype, implying a reduction in the density and total number of NeuN+ neurons. Scale bars: 100 µm, 5 µm. Post-hoc LSD tests:</p><p>(*<i>p</i><.05): significant difference between the stressed group vs. its control;</p><p>(#<i>p</i><.05): significant difference between the control NULL vs. the control WT mice.</p

Spatial memory and exploratory and anxiety-like behaviour on the hole-board test.

<p>Mean values ± SEM. (A) Chronic stress impaired reference memory in both genotypes, but the deficit was more dramatic in maLPA₁-null mice. (B) This differential impairment was related to memory consolidation rather than acquisition, although acquisition was also affected by stress. (C) Chronic stress did not impair working memory, but null groups performed worse on the first training day. (D) MaLPA₁-null mice spent more time in the periphery (thigmotaxis) throughout the testing procedure. Only WT mice showed hyperactivity (locomotion, head dipping) in response to stress. D: Training day. Post-hoc LSD tests: (*<i>p</i><.05; **<i>p</i><.001): significant difference between the stressed group vs. its control; (#<i>p</i><.05; ##<i>p</i><.001): significant difference between the control NULL vs. the control WT mice.</p

c-Fos immunohistochemistry in the rat dorsal periaqueductal gray matter (PAG) following LPA 18∶1 infusion at doses of 0 and 2 µg.

<p>Analyses were performed in the dorsomedial and dorsolateral divisions of the PAG (<b>A</b>). Stereological quantification of c-Fos immunoreactive (IR) nuclei in the DPAG (<b>B</b>). Low magnification microphotographs for c-Fos immunohistochemistry are depicted for the vehicle (<b>C</b>)- and LPA 18∶1 (<b>D</b>)-treated groups. In addition, high magnification images for the vehicle (<b>E</b>)- and LPA 18∶1 (<b>F</b>)-treated groups are shown. Each point represents the total number of immunopositive nuclei per animal. The dotted lines are medians (n = 4 animals pr group). The data were analyzed using Kruskal-Wallis one-way ANOVA. *p<0.05 denotes significant differences versus the vehicle-treated group, determined using Dunn’s <i>post-hoc</i> test. The arrowheads indicate immunopositive nuclei for c-Fos.</p

Rats were studied for novelty recognition in the Y maze (YMZ).

<p>LPA 18∶1 infusion at doses of 0, 0.4 and 2 <b>µ</b>g was carried out 5 min before the sample trial, and the test trial was performed 2 h later. The percent of rats of each group that first entered the novel arm (A), the total time of novel arm exploration (B) and the total arm entries (C) in the test trial were avaluated. The bars are the means ± SEM (n = 11–12 animals per group). The data were analyzed using a Chi-square test (A) or an one-way ANOVA followed by Bonferroni’s <i>post-hoc</i> tests (B–C). *p<0.05 denote significant differences versus the vehicle-treated group. The comparison of the 2 <b>µ</b>g and the vehicle group in (B) was significant at p = 0.0558.</p

Time schedules and experimental designs.

<p>Male Wistar rats were sham injected at least two times prior to LPA treatment to familiarize animals with the i.c.v. procedure. The open field test (OFT) and elevated plus maze (EPM) were performed under novelty (A) and habituation (B) conditions after 5 min of LPA 18∶1 infusion. For habituation, the rats were sham injected and tested in both paradigms 24 h before LPA infusion. Rats were exposed to the sample trial of the Y maze (YMT) 5 min after LPA 18∶1 administration, and performed the test trial 2 h later (C). The forced swimming test (FST) was performed after 5 min of LPA 18∶1 infusion (D). Food and water intake were evaluated at different times in 24 h food-deprived animals after 5 min of LPA 18∶1 infusion (E). c-Fos immunoreactivity (IR) was performed through perfusion after 90 min of LPA 18∶1 infusion (F).</p

Wistar rats were studied in the elevated plus maze (EPM) and open field test (OFT) under novelty and habituation conditions following LPA 18∶1 infusion at doses of 0, 0.4 and 2 µg.

<p>In the EPM, the time (s) exploring the exposed arms (A) and the total number of arm entries (B) were evaluated under novelty conditions. Similarly to the novelty conditions, the time exploring the open arms (C) and the total arm entries (D) were evaluated in animals previously habituated to the EPM (A). Locomotor activity was measured based on the number of crossings (E) and the time (%) spent in the center of the field (F) under novelty conditions. Again, both the number of crossings (G) and the percentage of time spent in the center (H) were evaluated under habituation conditions. The bars are the means ± SEM (n = 7–12 animals per group). The data were analyzed using one-way ANOVA. *p<0.05 and ***p<0.001 denote significant differences versus the vehicle-treated group, determined using Bonferroni’s <i>post-hoc</i> test.</p

Time-course effects of i.c.v. administration of LPA 18∶1 or vehicle on food and water intake in 24 h food-deprived Wistar rats.

<p>¹ Data are means ± SEM of 8 determinations per group.</p