Selective p38α MAPK deletion in serotonergic neurons produces stress resilience in models of depression and addiction

Abstract

Maladaptive responses to stress adversely affect human behavior, yet the signaling mechanisms underlying stress-responsive behaviors remain poorly understood. Using a conditional gene knockout approach, the α isoform of p38 mitogen-activated protein kinase (MAPK) was selectively inactivated by AAV1-Cre-recombinase infection in specific brain regions or by promoter-driven excision of p38α MAPK in serotonergic neurons (by Slc6a4-Cre or ePet1-Cre) or astrocytes (by Gfap-CreERT2). Social defeat stress produced social avoidance (a model of depression-like behaviors) and reinstatement of cocaine preference (a measure of addiction risk) in wild-type mice, but not in mice having p38α MAPK selectively deleted in serotonin-producing neurons of the dorsal raphe nucleus. Stress-induced activation of p38α MAPK translocated the serotonin transporter to the plasma membrane and increased the rate of transmitter uptake at serotonergic nerve terminals. These findings suggest that stress initiates a cascade of molecular and cellular events in which p38α MAPK induces a hyposerotonergic state underlying depression-like and drug-seeking behaviors.

Figure 1. p38α expression in the dorsal raphe nucleus is required for stress behavior

(A) Representative low power immunofluorescence images of social defeat stress induced pp38-ir (green) in TPH-ir cells (red) of the DRN. (A¹) Quantification ±SEM of pp38-ir in DRN from unstressed (naïve), social defeat stress (SDS) and social defeated stress exposed norBNI (10 mg/kg, i.p.) injected mice (**p < 0.01, SDS vs. Naïve). Inset, representative black and white low power immunofluorescence images of social defeat stress induced pp38-ir, scale bar = 200μm (B) Schematic of AAV1 induced cre-recombination of the floxed p38α MAPK allele and STOP sequence controlling Rosa26^YFP gene expression. (C) Representative images of pp38-ir (red) and YFP (green) fluorescence following AAV1-Cre-GFP or AAV1-ΔCre-GFP injection into the DRN. Mice were pretreated with KOR agonist (U50,488, 20 mg/kg, i.p, 20 min prior to perfusion). Images show that AAV-cre expressing cells lack pp38-ir, confirming effective localized DRN p38α deletion in cells where Cre-activity also promoted YFP expression by the Rosa reporter. (D) Conditioning procedure for SDS induced reinstatement of cocaine seeking. (E) Cocaine place preference scores, calculated as post-test minus pre-test on the cocaine-paired side, and SDS-induced reinstatement scores of extinguished place preference in DRN-injected animals (n = 5–8; *, P < 0.05 t-test compared to AAV1-ΔCre). Bars represent means± SEM. (F) Preference scores (mean ± SEM) for conditioned place aversion to kappa opioid agonist U50,488 (2.5 mg/kg, i.p) from mice injected with either AAV1cre-GFP or AAV1Δcre-GFP into their DRN or Nucleus Accumbens (NAc) (*p < 0.05, AAV1cre-GFP vs. AAV1Δcre-GFP; n = 8). See also Figure S1.

Figure 2

(A) Schematic of cell type specific p38α deletion. Floxed p38α and ROSA^YFP reporter mice were crossed to mice expressing Cre-recombinase under the control of Pet1, serotonin transporter, or the tamoxifen inducible glial fibrillary acidic protein (GFAP) Cre^ERT2 transgene. Representative images showing TPH-ir and YFP in p38αCKO^ePet mice (B), p38αCKO^SERT (C), and p38αCKO^GFAP (D) mice. Insets show higher power images with arrows directed towards yellow cells indicating overlap of TPH/YFP expression. Representative images showing TPH and p38α-ir in wild type (E), p38αCKO^ePet (F), and p38αCKO^SERT. (G) Representative images from wild type mice showing the absence of phosphorylated p38 MAPK (pp38-ir) following saline treatment (H) and increased pp38-ir following treatment with U50,488 20 mg/kg, i.p., 20 min prior) (I). Insets show intact TPH labeling in the same fields. (J) Quantitation of p38α-ir in TPH positive cells in the dorsal raphe nucleus. Data show a significant reduction in p38α expression in both p38α CKO^ePET and p38α CKO^SERT mice (***p < 0.001, ANOVA, Bonferroni). (K). Representative images from in p38α CKO^ePET mice showing the absence of pp38-ir following saline treatment (K) and following treatment with U50,488 (L). Insets show intact TPH staining. (M) Quantitation of p38α-ir expressed in TPH-negative cells in the DRN. Data are representative of 4–8 animals per group. See also Figure S3.

Figure 3. Negative affective behavior requires expression of p38α in serotonergic neurons

(A) Representative traces of mouse locomotion (red lines) in unstressed and social defeat stressed wild type or mice lacking p38α in serotonergic neurons (p38α CKO^ePet). Data show that SDS caused mice to retreat to Zone 2 or 3 (Z2,3, far corners). Mice pretreated with norBNI (10 mg/kg, i.p., 24 hr prior) or with serotonergic p38α deletion (p38αCKO^ePet) show normal exploration of the interaction zone (IZ). (B). Quantification of social interaction scores in mice following SDS. Dashed line represents the social interaction scores for unstressed mice (n=8, * p < 0.05 vs. control saline or p38α^Δ/lox, t-test). (C) Place Preference scores following conditioning with U50,488 (2.5 mg/kg) in wild type, p38α^Δ/lox and p38αCKO^ePet mice (n = 8–10, ANOVA, p < 0.05 vs. control). (D) Place Preference scores (means± SEM following conditioning with U50,488 (2.5 mg/kg) in p38α wild type vs. p38α ^Δ/lox and p38α CKO^SERT mice (n = 8–10, ANOVA, p < 0.05 vs. control). (E) Place Preference scores ± SEM following conditioning with U50,488 (2.5mg/kg) in wild type or p38αCKO^GFAP mice (n = 6–8). (F) Swim-stress induced immobility scores for wild type mice, p38α^Δ/lox, or p38αCKO^SERT (Data are means ± SEM; ANOVA, * p < 0.01, n = 6–8). See also Figure S3.

Figure 4. Disruption of p38α in serotonergic neurons protects against SDS-induced reinstatement of drug seeking

(A) Representative images of SDS induced phospho-p38-ir in each mouse line. Data show an absence of SDS-induced pp38-ir in TPH-ir cells in both p38αCKO^SERT and p38αCKO^ePet mice, but show an intact increase in pp38-ir in TPH-ir cells of p38α CKO^GFAP mice. (Pixel intensities of pp38-ir were quantified from these and replicate images and shown in Supplemental Figure 3J. SDS did not significantly increase pp38-ir in DRN of p38αCKO^ePet or p38αCKO^SERT; whereas pp38-ir was significantly increased in DRN of p38αCKO^GFAP mice. (B) Mouse place preference scores (± SEM) following cocaine (15 mg/kg, s.c.) conditioning (C) Mouse place preference scores after extinction (± SEM) and following social defeat (p < 0.05, ANOVA, Bonferroni post hoc). (D) Mouse place preference scores following extinction then cocaine priming (15 mg/kg, s.c.) (n = 8–20). See also Figure S4 for additional behavioral characterization.

Figure 5. Investigation of 5HT uptake by SERT

(A) Place preference scores (± SEM) following conditioning of wild type mice treated either with U50,488 (2.5 mg/kg) (U50/Saline), with the selective SERT reuptake inhibitor citalopram (CPM) (15 mg/kg, i.p., 30 min prior to U50,488) (U50/CPM), or with citalopram alone (Saline/CPM). Citalopram prior to KOR agonist significantly blocked U50,488 CPA (ANOVA, p < 0.05, n =8–10) (B,C). Representative RDEV traces of 5-HT uptake from paroxetine (red traces) and non-paroxetine (black traces) treated synaptosomes isolated from control (B) or U50,488 (2.5 mg/kg, i.p. x2) treated animals (C). Note the larger difference in slope for U50,488 treated than control animals. (D) Administration of U50,488 (2.5 mg/kg, i.p. x2 24hr apart) to mice, 30 min prior to synaptosomal isolation, increased 5-HT uptake by SERT compared to saline treated controls (n = 10–16, * P < 0.01). This effect of U50,488 was blocked by pretreatment of the mice with norBNI (10 mg/kg). (E) Administration of U50,488 (2.5 mg/kg, i.p., x2), increased serotonin uptake by SERT in synaptosomes generated from p38α^+/+, p38α^Δ/lox, and p38α_+/+,SERTcre mice, but not from p38αCKO^SERT mice (n =10–16, * p < 0.05). (F) Administration of U50,488 (2.5 mg/kg, i.p. x2) 30 min prior to preparation of synaptosomes, did not significantly increase serotonin uptake by the low-affinity transporters (n=10–16).

Figure 6. p38α MAPK is required for social defeat stress induced cell surface SERT trafficking

(A) Representative immunoblot of total SERT levels in the different mouse lines used in this study. Data show both species of SERT (75 and 98 kDa) are present in these strains and the absence of SERT-ir in the Slc6a4 knockout (SERT-KO) mouse. Actin-ir was used to control for protein loading. (B). Representative immunoblot of surface SERT expression in biotinylated synaptosomes isolated from unstressed mice (no stress), from mice after SDS, and from mice pretreated with norBNI (10 mg/kg) 24 hr prior to SDS. (Anti-streptavidin-ir confirms equal protein loading after biotinylation and pull down. (C) Quantification of SERT-ir surface expression following SDS of saline-treated wild type, norBNI-treated wild type, and p38αCKO^ePet mice. (* p < 0.05, ** p< 0,01, *** P< 0.001, ANOVA, Bonferroni post-hoc) (D). Quantification of SERT-ir surface expression following U50,488 treatment of wild type and p38α CKO^ePet mice. (*p < 0.05, t-test). n= 8–10 in replicate, and each was taken from a separate animal (E) Cartoon model depicting p38α MAPK dependent SERT translocation and decreased extracellular 5HT. See also Figure S5.