Figure 1.
Time schedules and experimental designs.
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).
Figure 2.
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.
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 post-hoc test.
Figure 3.
Rats were studied for novelty recognition in the Y maze (YMZ).
LPA 18∶1 infusion at doses of 0, 0.4 and 2 µg 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 post-hoc tests (B–C). *p<0.05 denote significant differences versus the vehicle-treated group. The comparison of the 2 µg and the vehicle group in (B) was significant at p = 0.0558.
Figure 4.
Wistar rats were studied in the forced swimming test (FST) following LPA 18∶1 infusion at doses of 0, 0.4 and 2 µg.
The immobility time (s) was evaluated in these animals. The bars are the means ± SEM (n = 8 animals per group). The data were analyzed using one-way ANOVA. *p<0.05 and **p<0.01 denote significant differences versus the vehicle-treated group, determined using Bonferroni’s post-hoc test.
Table 1.
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.
Figure 5.
c-Fos immunohistochemistry in the rat dorsal periaqueductal gray matter (PAG) following LPA 18∶1 infusion at doses of 0 and 2 µg.
Analyses were performed in the dorsomedial and dorsolateral divisions of the PAG (A). Stereological quantification of c-Fos immunoreactive (IR) nuclei in the DPAG (B). Low magnification microphotographs for c-Fos immunohistochemistry are depicted for the vehicle (C)- and LPA 18∶1 (D)-treated groups. In addition, high magnification images for the vehicle (E)- and LPA 18∶1 (F)-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 post-hoc test. The arrowheads indicate immunopositive nuclei for c-Fos.
Figure 6.
Distribution of LPA1 receptor immunoreactivity (IR) in the periaqueductal gray matter (PAG) of adult rats and mice.
Coronal brain section showed the LPA1 receptor IR in the PAG of the adult Wistar rat (A). Representative micrographs from image A show that LPA1 IR is distributed in the dorsal (DPAG) (B) and lateral (LPAG) (C) area of rats. Comparable coronal sections of the PAG area in wild-type mice showed LPA1 IR in the DPAG and LPAG (D). Enlarged from image D, neuronal cell bodies and fibers display the expression of LPA1 (E). Magnified from image E, cell bodies and fibers are observed in detail (F). In maLPA1-null adult mice, LPA1 receptor IR is absent, and only the aqueductal ependyma exhibits a non-specific reaction (G). The arrowheads and asterisks indicate cell bodies and fibers, respectively. Scale bars: A, D, G, 500 µm; B, C, E, 180 µm; F, 50 µm.