DAT isn't all that: cocaine reward and reinforcement require Toll-like receptor 4 signaling

Abstract

The initial reinforcing properties of drugs of abuse, such as cocaine, are largely attributed to their ability to activate the mesolimbic dopamine system. Resulting increases in extracellular dopamine in the nucleus accumbens (NAc) are traditionally thought to result from cocaine's ability to block dopamine transporters (DATs). Here we demonstrate that cocaine also interacts with the immunosurveillance receptor complex, Toll-like receptor 4 (TLR4), on microglial cells to initiate central innate immune signaling. Disruption of cocaine signaling at TLR4 suppresses cocaine-induced extracellular dopamine in the NAc, as well as cocaine conditioned place preference and cocaine self-administration. These results provide a novel understanding of the neurobiological mechanisms underlying cocaine reward/reinforcement that includes a critical role for central immune signaling, and offer a new target for medication development for cocaine abuse treatment.

Figure 1. Computer in silico modeling of cocaine interactions with the TLR4/MD-2 receptor complex

(a) Crystalline structure of TLR4 (green) and MD2 (blue). (b) TLR4 (green) and MD2 (blue), the brown fill is the location that cocaine, and TLR4 antagonists (+)-naloxone and (+)-naltrexone prefer to dock. (c) Magnified image of the MD2 structure (blue) and preferred docking location of cocaine (red), (+)-naltrexone (green), and (+)-naloxone (yellow). Images are compositions of individual docking simulations superimposed and indicate that each compound prefers to dock in the same location. Cocaine docking in the presence (+)-naloxone and (+)-naltrexone causes a fundamental shift in docking conformation.

Figure 2. Biophysical characterization of cocaine and MD-2 binding

(a) Schematic illustration. Biotin labeled cocaine aptamer was immobilized onto the streptavidin coated plate as the probe for capturing cocaine. Cocaine bound MD-2 was detected by IgG-horseradish peroxidase (HRP) conjugate. It should be noted that the illustration is only a graphic presentation and does not represent the scale of each component. (b) Human MD2 (10 µg/mL) bound varying concentrations of cocaine against 10 µM of biotin labeled cocaine aptamer in a concentration-dependent manner; negative control protein A binding was negligible. c) Varying concentrations of human MD-2 bound cocaine (4 µM) in a concentration-depend manner against a fixed concentration of cocaine-aptamer (4 µM); the negative control protein, BSA, demonstrated negligible binding. (d) LPS concentration-dependently displaced human MD2 (40 µg) binding to cocaine (4 µg) against cocaine aptamer (4µg). (e) Florescent competitive binding assay: Bis-ANS fluorescent signaling increases upon MD-2 binding, cocaine decreased Bis-ANS fluorescence in a concentration-dependent manner, indicating that it competitively binds MD2. Data fitting to a one-site competitive model yields a K_i of 23.9 ± 5.9 µM.

Figure 3. Cocaine-induced signaling in isolated neonatal microglial cells is TLR4 dependent

a) LPS dose dependently upregulates mRNA expression of IL-1β (p < 0.01 and p <0.001, bonferroni post hoc) in neonatal microglia following 1 hr incubation (one-way ANOVA, F_(5,23) = 62.29,p < 0.003). b) Cocaine (0.1 µM and 1µM) upregulates mRNA expression of IL-1β (p < 0.01, bonferroni post hoc) in neonatal microglia following a 1 hr incubation period (one-way ANOVA, F_(4,19) = 11.56, p = 0.0002). c) (+)-Naloxone (1, 10, 100 µM) suppresses cocaine-induced upregulation of IL-1β mRNA (p < 0.0001; two-way ANOVA, interaction F_(6,33) = 19.68, p < 0.0001, bonferroni post hocs). Incubation with (+)-naloxone alone had no effect on IL-1β mRNA expression (p > 0.05).

Figure 4. (+)-Naloxone suppresses cocaine-induced upregulation of interleukin-1β mRNA in the VTA

(a) Cocaine (10 mg/kg, i.p.) induced upregulation of IL1β mRNA in the VTA (p < **0.01) but not the NAc or vmPFC. Two-way ANOVA revealed an effect of region (p = 0.034) and time following cocaine injection (p = 0.017), data are means ± SEMs, n = 5–6/group. (b) Cocaine-induced (10 mg/kg, i.p.) upregulation compared to saline (p = 0.01) of IL1β mRNA within the VTA is attenuated by (+)-naloxone (2.5 mg/kg given in two s.c. injections, with the first injection 10 min prior to the second which is paired with cocaine) administration (p <0.05). Bonferroni post-hocs were preceded by two-way ANOVA, indicating a main effects of cocaine (F_(1,17)=4.61, p=0.001) and (+)-naloxone (F_(1,17)=4.61, p=0.0447). Data are means ± SEMs, n=5–6/group. (c) Cocaine-induced (10 mg/kg, i.p.) upregulation compared to saline (p < 0.01) of IL1β mRNA within the VTA is attenuated by (+)-naloxone (2.5 mg/kg given in two s.c. injections, with the first injection 10 min prior to the second which is paired with cocaine) administration (p < 0.01). Bonferroni post-hocs were preceded by two-way ANOVA, indicating main effects of cocaine (F_(1,16)=5.18, p=0.03) and (+)-naloxone (F_(1,16)=16.12, p=0.001). Data are means ± SEMs, n=5/group.

Figure 5. Cocaine induced increases of NAc dopamine are dependent on TLR4 and IL-1β signaling within the VTA

(a) Cocaine (10 mg/kg, s.c.) produces elevated extracellular dopamine in the NAc 40 min (p < 0.0001) and 60 min (p < 0.01) following drug administration (repeated measures two-way ANOVA with bonferroni post-hocs, time and treatment interaction p < 0.001). Administration of (+)-naloxone (2.5 mg/kg in two s.c. injections spaced 10 min apart) blocked this effect; there were no differences between this group and the saline or (+)-naloxone treated rats. Prior to drug treatment, there were no differences in extracellular dopamine concentrations in the NAc shell across all groups. Data are means ± SEMs; n = 4/group. (b) Cocaine (10 mg/kg, i.p.) produces elevated extracellular dopamine in the NAc that sustains for 40–100 min (bonferroni, ****p < 0.00001, **p < 0.01, *p <0.05) following drug administration. Intra-VTA LPS-RS blocked this effect. There were no differences between other treatment groups. Data are means ± SEMs; n = 4/group. (c) Cocaine (10 mg/kg, i.p.) produces elevated extracellular dopamine in the NAc that are significant at 40, 60, and 100 min (bonferroni, ****p < 0.00001, **p < 0.01) following drug administration. Intra-VTA IL1ra suppressed this effect. There were no differences between other treatment groups. Data are means ± SEMs; n = 3–4/group. (d) LPS (10 ng in 1 µL) microinjected into the VTA produces increased extracellular dopamine within the NAc compared to vehicle microinjection-controls (two-way ANOVA, effect of treatment F_(1,144)= 35.83, p < 0.0001 and time F_(11,144) = 2.89, p = 0.0018) 40 min and 100 min post-microinjection (p < 0.05, bonferroni post-hoc). Data are means ± SEMs; n = 5–7/group.

Figure 6. Microglial TLR4 signaling is necessary for the expression of cocaine-induced CPP

(a) Cocaine (10 mg/kg, i.p.) produces place preference (p<0.0001) and (+)-naloxone (5 mg/kg divided into two s.c. injections spaced 10 min apart) suppresses this effect; no other treatment groups were different from the saline group, nor from one another. Interaction of cocaine and (+)-naloxone (F_(1,27) = 13.6, p = 0.001), with a main effect of each (n < 0.01), detected by two-way ANOVA followed by bonferroni, data are mean ± SEMs, n = 7–9/group. (b) Cocaine (10 mg/kg, i.p.) produces significant place preference (p < 0.05, two-way ANOVA with bonferroni). Minocycline (50 mg/kg, gavage, 12 h prior and 25 mg/kg 45 min prior to conditioning) significantly attenuated cocaine-induced (10mg/kg, i.p.) place preference. In the absence of cocaine, neither water nor minocycline altered place preference (main effect of cocaine (p < 0.01) and minocycline (p < 0.01), data are mean ± SEMs, n = 11–12/group).

Figure 7. TLR4 signaling is required for cocaine self-administration

(a) Response rates maintained by cocaine injections were affected (F_4,20 = 6.7, p = 0.001, one-way repeated measures ANOVA ) by dose. The highest rate of responding is maintained by cocaine at a dose of 0.27 mg/kg/injection (**p = 0.002). A pre-session dose of 22.4 mg/kg (+)-naltrexone (s.c.) decreases response rates maintained at 0.27 mg/kg/injection cocaine (***p<0.001). There is an interaction of cocaine dose and (+)-naltrexone dose (F_8,40 = 3.7, p = 0.003) and a main effect of cocaine dose (F_(4,40) = 8.3, p < 0.001). EXT: extinction; data are means ± SEMs; n = 6/group. (b) In contrast, food-maintained behavior was virtually insensitive to pre-session treatment with (+)-naltrexone (two-way repeated measures ANOVA, p = 0.909). EXT: extinction; data are means ± SEMs; n = 6/group). (c) (+)-Naltrexone was more potent in decreasing responding maintained by the maximal reinforcing doses of cocaine than in decreasing responding maintained by food presentations. Data are means ± SEMs; n = 6/group. (d) C3H/HeJ TLR4 mutant mice do not self-administer cocaine, while their normal C3H/FeJ normal TLR4 counterparts demonstrate normal cocaine self-administration. Repeated measures two-way ANOVA reveals an interaction of genotype and drug (p = 0.0134) and a main effect of both drug (p = 0.0143) and genotype (p = 0.0029). On days 2–7, FeJ mice self-infused more cocaine than HeJ TLR4 mutant mice, whereas HeJ mice demonstrated no difference in cocaine infusions than either FeJ or HeJ saline groups (**p <0.001, ***p<0.0001 bonferroni post-hoc). Data are ± SEMs; n = 6–12/group. (e) Over the 7 days of testing HeJ TLR4 mutant mice self-administer less cocaine (***p = 0.0002, t₍₁₇₎=4.821, unpaired t-test) and (f) nose-poked less (***p < 0.0001, t₍₁₆₎=5.421, unpaired t-test) than their FeJ normal TLR4 counterparts. Data are ± SEMs; n = 7–12/group. (g) TLR4 mutant C3H/HeJ mice self-administer sucrose no differently than their normal TLR4 C3H/FeJ counterparts. (h) There are no differences in the daily number of sucrose (10%) deliveries between C3H/FeJs and C3H/HeJs (two-way ANOVA, p = 0.80), (i) the total amount of sucrose earned over the 7 day testing period (two-tailed, unpaired t-test, p = 0.16), or (j) the final ratio of nose-pokes (two tailed, unpaired t-test, p = 0.44). All data are ± SEMs; n = 9/group.