Dorsal raphe neuroinflammation promotes dramatic behavioral stress dysregulation

Abstract

Impulsivity, risk-taking behavior, and elevated stress responsivity are prominent symptoms of mania, a behavioral state common to schizophrenia and bipolar disorder. Though inflammatory processes activated within the brain are involved in the pathophysiology of both disorders, the specific mechanisms by which neuroinflammation drives manic behavior are not well understood. Serotonin cell bodies originating within the dorsal raphe (DR) play a major role in the regulation of behavioral features characteristic of mania. Therefore, we hypothesized that the link between neuroinflammation and manic behavior may be mediated by actions on serotonergic neurocircuitry. To examine this, we induced local neuroinflammation in the DR by viral delivery of Cre recombinase into interleukin (IL)-1β(XAT) transgenic male and female mice, resulting in overexpressing of the proinflammatory cytokine, IL-1β. For assertion of brain-region specificity of these outcomes, the prefrontal cortex (PFC), as a downstream target of DR serotonergic projections, was also infused. Inflammation within the DR, but not the PFC, resulted in a profound display of manic-like behavior, characterized by increased stress-induced locomotion and responsivity, and reduced risk-aversion/fearfulness. Microarray analysis of the DR revealed a dramatic increase in immune-related genes, and dysregulation of genes important in GABAergic, glutamatergic, and serotonergic neurotransmission. Behavioral and physiological changes were driven by a loss of serotonergic neurons and reduced output as measured by high-performance liquid chromatography, demonstrating inflammation-induced serotonergic hypofunction. Behavioral changes were rescued by acute selective serotonin reuptake inhibitor treatment, supporting the hypothesis that serotonin dysregulation stemming from neuroinflammation in the DR underlies manic-like behaviors.

Keywords: dorsal raphe; interleukin-1β; mania; neuroinflammation; serotonin; stress.

Figure 1.

IL-1β overexpression within the DR causes local neuroinflammation. A, Location of the DR in the Mouse Brain Atlas (Paxinos and Franklin, 2001). B, C, Representative images highlight spatially restricted β-Gal (B) and GFP immunofluorescence (C) following AAV-Cre infusion. b', c', Higher magnification of subfield. D, AAV-Cre infusion significantly increases IL-1β mRNA in both males and females in the DR. E–M, Immunofluorescent characterization of microglia (E, CD11b) neurons (F, J, NeuN), astrocytes [G, K, GFAP; H, L, aquaporin-4 (AQP4)] and IDO (I, M), an enzyme that catabolizes tryptophan in AAV-GFP-infused mice (E–H) and AAV-Cre-infused mice (I–L). N, ELISA of plasma samples reveals no effect of DR AAV-Cre infusion on circulating IL-1β. Data are presented as mean ± SEM, n = 6–7; **p < 0.01 denotes main effect of treatment following two-way ANOVA comparing sex (male vs female) or genotype (WT vs IL-1β) by treatment (AAV-GFP vs AAV-Cre).

Figure 2.

DR neuroinflammation causes manic-like behavior. A–F, In the elevated plus-maze, both male (A–C) and female (D–F) IL-1β^XAT mice infused with AAV-Cre display increased locomotion (A, D), time in the open arm (B, E), and time in the distal ends of the open arm (C, F). G–L, In the light/dark box, both male (G–I) and female (J–L) IL-1β^XAT mice infused with AAV-Cre travel a greater distance in the light compartment (distance, normalized to time spent in light compartment; G, J), transition more between light and dark compartments (H, K), and spend more time in the light compartment (I, L). M–P, In the locobox, male IL-1β^XAT mice infused with AAV-Cre travel more throughout the test (M) at a greater speed (N), while transitioning fewer times between compartments (O) and having no significant preference for either compartment (P). Q–T, Female mice showed a similar pattern as the males. Data are presented as mean ± SEM, n = 6–10; *p < 0.05, **p < 0.01 using within sex two-way ANOVA of genotype (WT vs IL-1β^XAT) by treatment (AAV-GFP vs AAV-Cre), followed by Tukey's multiple-comparison test.

Figure 3.

DR neuroinflammation increases male stress responsivity. A, Male IL-1β^XAT mice infused with AAV-Cre display an increase in corticosterone production in response to a 15 min restraint stress (shaded), while exhibiting no difference in baseline or recovery. B, No significant effects were observed in females. Data are presented as mean ± SEM, n = 9–11, **p < 0.001 using a within-sex two-way repeated-measures ANOVA comparing genotype (WT vs IL-1β) vs treatment (AAV-GFP vs AAV-Cre), with time as a repeated measure. Grey bar represents period of 15 min acute restraint stress.

Figure 4.

Acute microinfusion of IL-1β into the DR results in increased locomotion but no change in open-arm exploration in the elevated plus-maze. A–C, Male and female mice infused with IL-1β 30 min before testing on the elevated plus-maze display increased locomotor activity (A), but no change in open-arm exploration (B). None of the animals traveled to the distal ends of the open arms following DR infusion. PFC neuroinflammation does not induce manic-like behavior or physiology. D–H, Male and female IL-1β^XAT mice infused with AAV-Cre showed no difference in behavior on the elevated plus-maze (D–F) or light/dark box (G, H). I, J, Neither males (I) nor females (J) display differences in corticosterone response to an acute 15 min restraint stress (shaded). Data are presented as mean ± SEM, n = 7–8. *p < 0.05 denotes main effect of infusion following a two-way ANOVA comparing sex versus treatment (AAV-GFP vs AAV-Cre).

Figure 5.

IL-1β-mediated neuroinflammation in the DR results in broad changes in gene expression. A, Heat map illustrating significant differences in gene expression between AAV-GFP-infused and AAV-Cre-infused male and female DR RNA from IL-1β^XAT mice, where data are expressed as relative levels within each gene, and both animals and genes are organized by hierarchical clustering. B, C, Genes that were either significantly upregulated (B) or significantly downregulated (C), as determined by an false discovery rate of 0.01, are ranked by enrichment score and organized by functional gene clusters identified using the Database for Annotation, Visualization, and Integrated Discovery bioinformatics database (DAVID). D, A separate analysis was performed on genes annotated with the gene ontology term (GO) “neur*,” which identified 26 genes that are downregulated in IL-1β animals infused with AAV-Cre.

Figure 6.

IL-1β-mediated neuroinflammation in the DR results in serotonergic hypofunction. A, B, Immunofluorescence of TPH-immunoreactivity was used to identify serotonergic neurons (red), counterstained with the nuclear stain DAPI (blue), of AAV-GFP-infused (A) and AAV-Cre-infused (B) DR 8 weeks following AAV infusion (n = 4). C, Quantification revealed fewer TPH-immunoreactive cells in AAV-Cre-infused animals. D–F, HPLC analysis of biogenic amines of the DR (D) revealed less NE (E) but no change in DA (F). G–J, In the PFC (G), AAV-Cre-infused animals had less 5-HT (H), increased levels of the metabolite (I, 5HIAA), and an overall increase in neurotransmitter turnover (J, 5HIAA/5-HT ratio). Data are presented as mean ± SEM, n = 6–7; **p < 0.01, ***p < 0.0001 following t test comparing AAV-GFP and AAV-Cre infusion.

Figure 7.

Acute increase in 5-HT ameliorates manic-like phenotype in the elevated plus-maze. IL-1β^XAT mice were infused with AAV-GFP or AAV-Cre into the DR and tested for 3 consecutive weeks on the elevated plus-maze 32 min after intraperitoneal injection with saline (Tests 1, 3) or 30 mg/kg citalopram (Test 2, shaded). A–C, Male mice infused with AAV-Cre show a significant attenuation of distance traveled (A), open-arm time (B), and distal open-arm entries (C) following citalopram treatment. D–F, Females show a similar pattern of behavior. Data are presented as mean ± SEM, n = 4–5, *p < 0.05, **p < 0.01 following two-way ANOVA of treatment by trial. Post hoc test compared citalopram to each saline treatment.