Sleep Disturbance Alters Cocaine-Induced Locomotor Activity: Involvement of Striatal Neuroimmune and Dopamine Signaling

Abstract

Sleep disorders have high comorbidity with drug addiction and function as major risk factors for developing drug addiction. Recent studies have indicated that both sleep disturbance (SD) and abused drugs could activate microglia, and that increased neuroinflammation plays a critical role in the pathogenesis of both diseases. Whether microglia are involved in the contribution of chronic SDs to drug addiction has never been explored. In this study, we employed a mouse model of sleep fragmentation (SF) with cocaine treatment and examined their locomotor activities, as well as neuroinflammation levels and dopamine signaling in the striatum, to assess their interaction. We also included mice with, or without, SF that underwent cocaine withdrawal and challenge. Our results showed that SF significantly blunted cocaine-induced locomotor stimulation while having marginal effects on locomotor activity of mice with saline injections. Meanwhile, SF modulated the effects of cocaine on neuroimmune signaling in the striatum and in ex vivo isolated microglia. We did not observe differences in dopamine signaling in the striatum among treatment groups. In mice exposed to cocaine and later withdrawal, SF reduced locomotor sensitivity and also modulated neuroimmune and dopamine signaling in the striatum. Taken together, our results suggested that SF was capable of blunting cocaine-induced psychoactive effects through modulating neuroimmune and dopamine signaling. We hypothesize that SF could affect neuroimmune and dopamine signaling in the brain reward circuitry, which might mediate the linkage between sleep disorders and drug addiction.

Keywords: cocaine; dopamine; drug addiction; microglia; neuroinflammation; sleep fragmentation.

Figure 1

The Effects of SF on cocaine-mediated behavioral changes. (A) Schematic for the experimental design. (B) Cocaine significantly increased locomotor activity while SF partially blocked this hyperlocomotion (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 8–10). (C) Cocaine significantly increased the velocity while SF partially blocked this upregulation (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 8–10). (D) Cocaine significantly decreased the immobility time and SF partially blocked this downregulation (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 8–10). (E) SF decreased the distance travelled on the first and second day of cociane injection (15 mg/kg) during the 7-day injecition period (# p < 0.05, SF + cocaine vs. sham + cocaine). (F) SF decreased the distance travelled throughout the whole 7-day cocaine injection period (10 mg/kg) # p < 0.05, SF + cocaine vs. sham + cocaine). (G) Cocaine and SF increased the pecentage time in central area (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 8–10). (H) SF decreased the latency time of mice in the coordination test (* p < 0.05, vs. sham + saline).

Figure 2

SF blunted cocaine-mediated neuroinflammation in the striatum. (A) Cocaine increased IL1β levels and SF blocked the increase (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5). (B) There was no difference on IL6 levels across the four groups of mice (p = 0.3794). (C) There was no difference on TNFα levels groups of mice (p = 0.3373). (D) Cocaine increased CCL2 levels and SF blocked such upregulation mediated by cocaine (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5). (E) Cocaine decreased IL10 levels (* p < 0.05, vs. sham + saline). (F) There was no difference in TGFβ levels across groups of mice (p = 0.8469).

Figure 3

Combined effects of SF and cocaine on microglial immune responses. (A) Representative images for Iba1 immunostaining for adult microglia isolated from the striatum (scale bar = 50 μm). (B) The enrichment fold of TNFα, IL1β, IL6, and CCL2 in adult microglia comparing to striatal homogenates. (C) Cocaine increased IL1β levels in purified adult microglia and SF blocked upregulation mediated by cocaine (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5). (D) Cocaine and chronic SF together significantly increased IL6 levels in purified adult microglia (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5). (E) Cocaine and SF decreased TNFα levels in purified adult microglia (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5). (F) Cocaine and chronic SF together increase SDs CCL2 levels in purified adult microglia (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5). (G) Cocaine and SF together decreased IL10 levels in purified adult microglia (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5). (H) Cocaine and SF together increased TGFβ levels in purified adult microglia (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5).

Figure 4

Combined effects of SF and cocaine on astrocytes. (A) Cocaine increased the signal intensity of Iba1 signaling and SF blocked its upregulation. Left: representative images for Iba1 immunostaining (scale bar 10 μM); right: statistical analysis for Iba1 signaling in these four groups of mice (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5). (B) Cocaine and SF had no effects on the intensity of GFAP signaling. Left: representative images for GFAP immunostaining (scale bar 10 μM), right: statistical analysis for GFAP signaling in the four groups of mice (p > 0.05, n = 5). (C) Representative GFAP WBs image for the striatum across groups of mice; β-actin were served as a protein load control (p > 0.05).

Figure 5

Effects of cocaine and SF on NLRP3 inflammasome and dopamine signaling in vivo. (A) Representative WBs images for NLRP3, ASC, and caspase 1 in the striatum across groups of mice; β-actin served as a protein load control. (B) NLRP3 levels in the striatum across groups of mice (* p < 0.05, vs. sham + saline; # p < 0.05, SF + cocaine vs. sham + cocaine, n = 5). (C) ASC levels in the striatum across groups of mice (p > 0.05). (D) mCasp 1 levels in the striatum across groups of mice (* p < 0.05, vs. sham + saline). (E) Representative WBs images for DRD1, TH, DAT, NLRP3, ASC, and PSDs95 in the striatum across groups of mice, β-actin served as a protein load control. (F–I) Levels of DRD1, TH, DAT, and PSDs95 in the striatum across groups of mice (p > 0.05).

Figure 6

Effects of SF on cocaine-mediated locomotion sensitivity and neuroimmune signaling in withdrawn mice. (A) Schematic for the experimental design. (B) Challenging with cocaine significantly increased locomotor activity in both groups of mice (±SF, * p < 0.05). (C) The fold increase induced by cocaine in sham mice was significantly higher than in SF mice (* p < 0.05). (D) Challenging with cocaine significantly increased IL1β levels and SF blocked the upregulation (* p < 0.05, vs. sham + saline, # p < 0.05, vs. cocaine + sham). (E) Challenging with cocaine increased IL6 levels in both groups of mice (±SF) (* p < 0.05, vs. sham + saline). (F) There was no significant change in TNFα levels across the groups (p > 0.05). (G) Challenging with cocaine increased CCL2 levels in SF mice (* p < 0.05, vs. sham + saline). (H) Challenging with cocaine increased TGFβ levels in mice without SF but not in mice with SF (* p < 0.05, vs. sham + saline. (I) Challenging with cocaine increased IL10 levels in SF mice (* p < 0.05, vs. sham + saline).

Figure 7

Effects of SF and cocaine on microglial activation and dopamine system in the withdrawn mice. (A) Representative WBs images for CD11b and GFAP in the striatum of three groups of mice; β-actin served as a protein load control. (B) Challenging with cocaine significantly increased CD11b levels in chronic ±SF mice (* p < 0.05, vs. sham + saline). (C) Challenging with cocaine had no effects on GFAP levels across groups (p > 0.05). (D) Representative WBs images for ASC and Casp 1 in the striatum of three groups of mice; β-actin served as a protein load control. (E) Challenging with cocaine significantly increased ASC levels in chronic ±SF mice (* p < 0.05, vs. sham + saline). (F) Challenging with cocaine had no effects on mCasp 1 levels across groups (p > 0.05). (G) Representative WBs images for DRD1, TH, DAT, and PSDs95 in the striatum of three groups of mice; β-actin served as a protein load control. (H) Challenging with cocaine significantly increased DRD1 levels in chronic ±SF mice (* p < 0.05, vs. sham + saline). (I) Challenging with cocaine significantly increased TH levels and SF blocked such upregulation (* p < 0.05, vs. sham + saline, # p < 0.05, vs. sham + cocaine). (J) Challenging with cocaine significantly increased DAT levels and SF blocked such upregulation (* p < 0.05, vs. sham + saline, # p < 0.05, vs. sham + cocaine). (K) Challenging with cocaine significantly increased PSDs95 levels and SF blocked such upregulation (* p < 0.05, vs. sham + saline, # p < 0.05, vs. sham + cocaine).

Figure 8

The summarization of the effects of SF on mice with different cocaine regimens.