Pioglitazone attenuates behavioral and electrophysiological dysfunctions induced by two-hit model of schizophrenia in adult rodent offspring

Abstract

Maternal infection and stress exposure, especially during childhood and adolescence, have been implicated as risk factors for schizophrenia. Both insults induce an exacerbated inflammatory response, which could mediate disturbance of neurodevelopmental processes and, ultimately, malfunctioning of neural systems observed in this disorder. Thus, anti-inflammatory drugs, such as PPARγ agonists, may potentially be used to prevent the development of schizophrenia. Microglia culture was prepared from the offspring of saline or poly(I:C)-injected mice. The cells were pretreated with pioglitazone and then, stimulated by LPS. Proinflammatory mediators and phagocytic activity were measured. Also, pregnant rats were injected with saline or poly(I:C) on GD17. The offspring were subjected to footshock during adolescence and subsequently injected with pioglitazone or vehicle. At adulthood, behavior and dopaminergic activity were evaluated. Pioglitazone reduced proinflammatory mediators induced by poly(I:C) microglia stimulated by LPS without affecting their decreased phagocytic activity. The PPARγ agonist also prevented the emergence of social and cognitive impairments, as well as attenuated the increased number of spontaneously active dopamine neurons in the VTA, observed in both males and females from poly(I:C) and stress group. Therefore, pioglitazone could potentially prevent the emergence of the schizophrenia-like alterations induced by the two-hit model via reduction of microglial activation.

Keywords: Behavioral impairments; Dopamine; Maternal immune activation; PPARγ agonist; Schizophrenia; Stress.

Figure 1:

Schematic representation of experimental design. A) Effects of pioglitazone on microglial cells from saline or poly(I:C) offspring. B) Effects of combined MIA and peripubertal stress exposure on behavior and dopaminergic neuron activity. C) Effects of pioglitazone on behavioral alterations and dopaminergic neuron activity induced by the two-hit model. POLY: poly(I:C); SAL: saline; PIO: pioglitazone; VEH: vehicle; FC: flow cytometry; FS: footshock; SA: social approach test (PD65); NOR: novel object recognition test (PD66-67); VTA: ventral tegmental area.

Figure 2:

Effects of pioglitazone (PIO 10μM) on mRNA expression of inflammatory mediators induced by 6h of LPS stimulation in microglial cells from saline (SAL) or poly(I:C) offspring. A) Expression of Il1b. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,17= 41.67; p<0.05; in vitro treatment: F_2,17= 103.9; p<0.05; maternal treatment x in vitro treatment: F_2,17=13.9; p<0.05), followed by SNK test. B) Expression of Tnfa. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,17= 37.29; p<0.05; in vitro treatment: F_2,17= 136.1; p<0.05; maternal treatment x in vitro treatment: F_2,17= 16.36; p<0.05), followed by SNK test. C) Expression of Il6. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F^1,17= 54.23; p<0.05; in vitro treatment: F_2,17= 60.9; p<0.05; maternal treatment x in vitro treatment: F_2,17= 16.09; p<0.05), followed by SNK test. D) Expression of Nos2. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,17= 41.47; p<0.05; in vitro treatment: F_2,17= 33.89; p<0.05; maternal treatment x in vitro treatment: F_2,17= 12.17; p<0.05), followed by SNK test. E) Expression of Ifa1. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,17= 108.8; p<0.05; in vitro treatment: F_2,17= 24.15; p<0.05), followed by SNK test. F) Expression of Cx3cr1. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,17= 9.674; p<0.05; in vitro treatment: F_2,17= 155; p<0.05; maternal treatment x in vitro treatment: F_2,17= 36.9; p<0.05), followed by SNK test. G) Expression of Ppparg. Data presented as mean ± SEM, analyzed by two-way ANOVA (in vitro treatment: F_2,17= 110; p<0.05; maternal treatment x in vitro treatment: F_2,17= 9.383; p<0.05), followed by SNK test.

Figure 3:

Effects of treatment with pioglitazone (PIO, 10μM) on cytokine levels induced by LPS (1ng/mL) in the supernatant of microglial cells from saline (SAL) or poly(I:C) offspring. A) IL-1β levels induced by 24h of LPS stimulation. Data presented as mean ± SEM, analyzed by two-way ANOVA (in vitro treatment: F_2,24= 64.16; p<0.05; maternal treatment x in vitro treatment: F_2,24= 3.557; p<0.05), followed by SNK test. B) TNF-α levels induced by 24h of LPS stimulation. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,24= 37.1; p<0.05; in vitro treatment: F_2,24= 260.8; p<0.05; maternal treatment x in vitro treatment: F_2,24= 9.408; p<0.05), followed by SNK test. C) IL-6 levels induced by 24h of LPS stimulation. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,17= 14.15; p<0.05; in vitro treatment: F_2,17= 70.86; p<0.05; maternal treatment x in vitro treatment: F_2,17= 5.901; p<0.05), followed by SNK test. D) IL-10 levels induced by 24h of LPS stimulation. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,18= 356.2; p<0.05; in vitro treatment: F_2,18= 132.3; p<0.05; maternal treatment x in vitro treatment: F_2,18= 94.64; p<0.05), followed by SNK test.

Figure 4:

Effect of pioglitazone (PIO 10μM) on phagocytic activity induced by LPS stimulation in microglial cells from saline (SAL) or poly(I:C) offspring. A) Representative plots of each experimental group. B) Phagocytosis quantification. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,14= 24.67; p<0.05; in vitro treatment: F_2,14= 55.28; p<0.05), followed by SNK test.

Figure 5:

Effects of maternal treatment with poly(I:C) (0.5mg/kg) or saline (SAL) combined with peripubertal stress (S) on behavioral tests in the adult male (n= 9-12 animals/group) and female (n= 10-12 animals/group) rats. A) Social preference measured in the SA test in male offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,37= 14.52; p<0.05; stress: F_1,37= 11.04; p<0.05), followed by SNK test. B) Discrimination index calculated from the retention session of NOR test in male offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,37= 10.6; p<0.05; maternal treatment x stress: F_1,37= 4.803; p<0.05), followed by SNK test. C) Social preference measured in the SA test in female offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,41= 5.931; p<0.05; stress: F_1,41= 8.403; p<0.05), followed by SNK test. D) Discrimination index calculated from the retention session of NOR test in female offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA vias (maternal treatment x stress: F_1,41= 8.14; p<0.05), followed by SNK test.

Figure 6:

Effects of maternal treatment with poly(I:C) (0.5mg/kg) or saline (SAL) combined with peripubertal stress (S) on electrophysiological activity of VTA in the adult male (n= 9-12 animals/group) and female (n= 10-12 animals/group) rats. A) Number of spontaneously active DA neurons per electrode track recorded in the VTA of male offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (stress: F_1,37= 8.484; p<0.05; maternal treatment x stress: F_1,37= 8.216; p<0.05), followed by SNK test. B) Number of spontaneously active DA neurons per electrode track in each portion of VTA in male offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (central – stress: F_1,37= 9.604; p<0.05; lateral – maternal treatment: F_1,37= 5.171; p<0.05; stress: F_1,37= 5.243; p<0.05; maternal treatment x stress: F_1,37= 4.551; p<0.05), followed by SNK test. C) Number spontaneously active DA neurons per electrode track recorded in the VTA of female offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (stress: F_1,41= 8.35; p<0.05; maternal treatment x stress: F_1,41= 11.33; p<0.05), followed by SNK test. D) Number of spontaneously active DA neurons per electrode track in each portion of VTA in female offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (lateral – stresse: F_1,41= 7.627; p<0.05; maternal treatment x stress: F_1,41= 7.643; p<0.05), followed by SNK test.

Figure 7:

Effects of treatment with pioglitazone (PIO, 10mg/kg) or vehicle (VEH) on behavioral alterations induced by two-hit model in the adult male (n= 9-12 animals/group) and female (n= 7-11 animals/group) rats. A) Social preference measured in the SA test in male offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,38= 20.95; p<0.05; treatment: F_1,38= 12.57; p<0.05; maternal treatment x treatment: F_1,38= 21.5; p<0.05), followed by SNK test. B) Discrimination index calculated from the retention session of NOR test in male offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (treatment: F_1,38= 6.027; p<0.05; maternal treatment x treatment: F_1,38= 5.639; p<0.05), followed by SNK test. C) Social preference measured in the SA test in female offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,33= 14.9; p<0.05; treatment: F_1,33= 7.937; p<0.05; maternal treatment x treatment: F_1,33= 34.27; p<0.05), followed by SNK test. D) Discrimination index calculated from the retention session of NOR test in female offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,33= 5.668; p<0.05; maternal treatment x treatment: F_1,33= 4.381; p<0.05), followed by SNK test.

Figure 8:

Effects of treatment with pioglitazone (PIO, 10mg/kg) or vehicle (VEH) on electrophysiological alterations induced by two-hit model in the adult male (n= 9-12 animals/group) and female (n= 7-11 animals/group) rats. A) Number of spontaneously active DA neurons per electrode track recorded in the VTA of male offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,38= 6.832; p<0.05; treatment: F_1,38= 13.27; p<0.05; maternal treatment x treatment: F_1,38= 9.111; p<0.05), followed by SNK test. B) Number of spontaneously active DA neurons per electrode track in each portion of VTA in male offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (medial – treatment: F_1,38= 4.525; p<0.05; maternal treatment x treatment: F_1,38= 6.976; p<0.05; lateral – treatment: F_1,38= 14.5; p<0.05; maternal treatment x treatment: F_1.38= 4.591; p<0.05), followed by SNK test. C) Number of spontaneously active DA neurons per electrode track recorded in the VTA of female offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (maternal treatment: F_1,33= 22.09; p<0.05; treatment: F_1,33= 8.415; p<0.05; maternal treatment x treatment: F_1,33= 17.59; p<0.05), followed by SNK test. D) Number of spontaneously active DA neurons per electrode track in each portion of VTA in female offspring. Data presented as mean ± SEM, analyzed by two-way ANOVA (medial – maternal treatment: F_1,33= 18.66; p<0.05; central – maternal treatment: F_1,33= 6.517; p<0.05; maternal treatment x treatment: F_1,33= 7.131; p<0.05; lateral – maternal treatment: F_1,33= 4.402; p<0.05; maternal treatment x treatment: F_1,33= 11.2; p<0.05), followed by SNK test.