Repeated administration of cocaine results in the development of behavioral sensitization, accompanied by a decrease in excitatory synaptic strength in the nucleus accumbens (NAc) through an unknown mechanism. Furthermore, glial cells in the NAc are activated by drugs of abuse, but the contribution of glia to the development of addictive behaviors is unknown. Tumor necrosis factor alpha (TNF-α), an inflammatory cytokine released by activated glia, can drive the internalization of synaptic AMPA receptors on striatal medium spiny neurons. Here we show that repeated administration of cocaine activates striatal microglia and induces TNF-α production, which in turn depresses glutamatergic synaptic strength in the NAc core and limits the development of behavioral sensitization. Critically, following a period of abstinence, a weak TLR4 agonist can reactivate microglia, increase TNF-α production, depress striatal synaptic strength, and suppress cocaine-induced sensitization. Thus, cytokine signaling from microglia can regulate both the induction and expression of drug-induced behaviors.

Recently, we have shown that TNF-α drives internalization of AMPARs on MSNs, reducing corticostriatal synaptic strength, and reduces the aberrant changes in striatal circuit function induced by chronic blockade of D2 dopamine receptors (). Glia are the main source of TNF-α in the CNS, and both microglia () and astrocytes () are activated by psychostimulants. Further, glia have been suggested to regulate drug-induced behavior (). Thus, glia through the release of TNF-α could have a mitigating effect on the circuit changes induced by cocaine. Here we demonstrate that striatal microglia are activated by cocaine, and moderate the synaptic and behavioral changes induced by the repeated administration of cocaine.

Changes in striatal processing, particularly in the NAc, are thought to be necessary for the maintenance of addictive behaviors, and repeated exposure to drugs of abuse leads to predictable changes in synaptic strength in the NAc (). Drugs of abuse, such as cocaine, elevate dopamine levels in the striatum, and ex vivo treatment of striatal medium spiny neurons (MSNs) with D1 dopamine receptor agonists or with cocaine increases the phosphorylation and insertion of AMPA receptors (). However, repeated cocaine treatment in vivo (5 days of noncontingent administration) results in an initial decrease in the AMPA/NMDA ratio on MSNs in the NAc, as measured 24 hr after the last cocaine injection (). A period of abstinence results in a gradual elevation of AMPA/NMDA ratios and AMPAR surface expression (), although a challenge dose of cocaine will result in lowered ratios and surface receptor content (). Self-administration of cocaine also causes similar changes in the NAc, with cocaine exposure causing a loss of AMPA receptors and depressing synaptic strength on MSNs and extended abstinence resulting in synaptic strengthening and accumulation of surface AMPA receptors (). This bidirectional plasticity suggests that other factors, in addition to dopamine, contribute to the synaptic changes induced by drug exposure.

Cell surface AMPA receptors in the rat nucleus accumbens increase during cocaine withdrawal but internalize after cocaine challenge in association with altered activation of mitogen-activated protein kinases.

Regulation of phosphorylation of the GluR1 AMPA receptor in the neostriatum by dopamine and psychostimulants in vivo.

D1 dopamine receptor stimulation increases the rate of AMPA receptor insertion onto the surface of cultured nucleus accumbens neurons through a pathway dependent on protein kinase A.

Results

Kalivas, 2009 Kalivas P.W. The glutamate homeostasis hypothesis of addiction. Figure 1 Cocaine Increases TNF-α Levels in the Nucleus Accumbens, which Causes Synaptic Depression on D1-MSNs and Antagonizes Cocaine-Induced Behavioral Sensitization Show full caption (A) Diagram of the time points used for experiments: 24 hr after a single injection of saline or cocaine (i.p. 15 mg/kg), 24 hr after five daily injections of saline or cocaine, and 10 days after five daily injections of saline or cocaine. (B) Representative confocal projection images of NAc immunostained for Iba1 (top) and TNF-α (bottom) from mice injected for 5 days with saline or cocaine (scale bar, 20 μm). (C) Five daily injections of cocaine increases TNF-α protein in the NAc. (D) Five daily injections of cocaine increases TNF-α mRNA in the ventral striatum. (E) Representative recording of EPSCs at −70 mV and +40 mV and mean AMPA/NMDA ratios from control slices and slices treated with 10 or 100 ng/ml TNF-α in D1 (red) and D2 (green) MSNs in the NAc core. AMPA/NMDA ratios were calculated using the peak amplitude at −70 mV for AMPA and the amplitude at +40 mV taken 40 ms after the peak at −70 mV. (F) Representative traces and mean AMPA/NMDA ratios from D1-MSNs in the NAc core, after 1 and 5 days of cocaine or saline in WT or TNF-α-KO mice. Ratios from mice injected with one or five daily doses of saline were not significantly different, and were combined. (G) Representative traces and mean AMPA/NMDA ratios from D1-MSNs in the NAc core from control slices or slices treated ex vivo with TNF-α. Treatment with 10 ng/ml TNF-α significantly reduced AMPA/NMDA ratios from TNF-α−/− mice treated 24 hr prior with cocaine. Ex vivo treatment with 100 ng/ml TNF-α did not further decrease AMPA/NMDA ratios from WT mice previously exposed to five daily cocaine injections. (H) Mean locomotor activity in response to cocaine injections in TNF-α−/− and WT mice, showing higher sensitization in TNF-α−/− mice, that was maintained after abstinence (n = 12 WT, 17 TNF-α−/− animals). (I) Blocking soluble TNF-α signaling only during the sensitization protocol (DN-TNF sensi) with DN-TNF is sufficient to sustain the elevation of the cocaine response to the challenge dose on day 15, while blocking TNF-α signaling during the withdrawal period (DN-TNF withd) had no effect on the response to the challenge dose after withdrawal (n = 16 DN-TNF sensi, 8 DN-TNF withd, 12 Control). Results are expressed as mean ± SEM, n (mice or cells) is given in bars. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. To determine the effect of in vivo cocaine exposure on TNF-α levels in the NAc, we measured TNF-α mRNA and protein levels in mice after i.p. injections of saline or cocaine. A single injection of cocaine had no effect on TNF-α levels (measured 24 hr postinjection), but 5 days of daily cocaine treatment (measured 24 hr after the final injection) increased both TNF-α mRNA and protein, compared to saline injected controls ( Figures 1 A–1D). TNF-α was no longer elevated following 10 days of abstinence from cocaine. To understand the impact of TNF-α on synaptic function in the NAc, we measured AMPA/NMDA ratios on MSNs in the NAc core. Alteration in NAc core AMPA receptors is involved in the expression of behavioral sensitization to psychostimulants ().

Lewitus et al., 2014 Lewitus G.M.

Pribiag H.

Duseja R.

St-Hilaire M.

Stellwagen D. An adaptive role of TNFα in the regulation of striatal synapses. We have previously shown that TNF-α drives internalization of AMPARs on MSNs in the dorsal striatum (). As repeated cocaine administration primarily affects direct-pathway MSNs, we tested specific subpopulations of MSNs in the NAc core for their response to TNF-α. Acute NAc slices were incubated with TNF-α and whole-cell recording made from Drd1a-td Tomato (D1) positive and negative (D2) MSNs. A low dose of TNF-α (10 ng/ml) had no significant effect on either cell type. However, 100 ng/ml TNF-α significantly reduced the AMPA/NMDA ratio on D1-MSNs, with a nonsignificant reduction in ratios on D2-MSNs ( Figure 1 E). These results suggest that D1-MSNs are more sensitive to TNF-α than D2-MSNs, although D2-MSNs may respond to a lesser degree.

Pascoli et al., 2012 Pascoli V.

Turiault M.

Lüscher C. Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour. −/− mice displayed an increased initial locomotor response to cocaine and increased sensitization, compared to WT mice (−/− mice ( Nakajima et al., 2004 Nakajima A.

Yamada K.

Nagai T.

Uchiyama T.

Miyamoto Y.

Mamiya T.

He J.

Nitta A.

Mizuno M.

Tran M.H.

et al. Role of tumor necrosis factor-alpha in methamphetamine-induced drug dependence and neurotoxicity. −/− mice on the first day of cocaine is not due to an acute loss of TNF-α signaling and is likely to be unrelated to the increase in sensitization. Depressing synaptic strength in the NAc can reduce behavioral sensitization to cocaine (). Behavioral sensitization is a simple model of drug-induced behavioral change, which measures the progressive increase in locomotor response to psychostimulants. TNF-αmice displayed an increased initial locomotor response to cocaine and increased sensitization, compared to WT mice ( Figure 1 H). This is similar to what has been observed with methamphetamine sensitization in TNF-αmice (). To exclude compensatory mechanisms resulting from the absence of TNF-α during development, we pharmacologically blocked the soluble form of TNF-α in WT mice using XENP1595 (a dominant-negative variant of TNF-α [DN-TNF]). WT mice were administered DN-TNF either during the 5 days of conditioning (to block TNF-α signaling during acquisition) or during the abstinence period starting immediately after the last cocaine injection (to test the role of TNF-α in the maintenance of the behavior). Blocking TNF-α signaling during acquisition was sufficient to increase sensitization as well as maintain the elevated response on the challenge day, while blocking TNF-α signaling during the 10 day period of abstinence had no effect on the response to the challenge dose ( Figure 1 I). These results suggest that TNF-α is active during acquisition but not during drug abstinence, consistent with the increased TNF-α expression observed during that period ( Figures 1 C and 1D). Further, the increased sensitivity observed in TNF-αmice on the first day of cocaine is not due to an acute loss of TNF-α signaling and is likely to be unrelated to the increase in sensitization.

Stellwagen and Malenka, 2006 Stellwagen D.

Malenka R.C. Synaptic scaling mediated by glial TNF-alpha. Figure 2 Microglia Are Activated by Cocaine and Release TNF-α to Antagonize Cocaine-Induced Behavioral Sensitization Show full caption (A) Mean locomotor activity in response to cocaine in mice that lack microglial TNF-α (CX3CR1-Cre+; TNF-αflox/flox) and littermate controls (CX3CR1-cre negative or TNF-α+/flox). The elevation was sustained for a final test dose of cocaine (n = 16 control; 12 microglia deletion). (B) Mean locomotor activity in response to cocaine injections in mice that lack astrocytic TNF-α (GFAP-Cre+; TNF-αflox/flox and littermate controls (GFAP-cre−; TNF-αflox/flox). GFAP-Cre mice had normal sensitization and response on the final test day (n = 25 per condition). (C) Purified microglia (CD11b+ fraction of cells) from whole striatum tissue express significantly more TNF-α mRNA compared to other cell types (CD11b− fraction) (five mice pooled per group in each experiment). (D) Five daily injections of cocaine increases TNF-α mRNA in striatal microglia (five mice pooled per group per experiment). (E) Representative confocal projection images of Iba1-labeled microglia in the NAc 24 hr after a single cocaine injection, five daily injections, or 10 days’ withdrawal (scale bar, 20μm). (F) Semiquantitative analysis of Iba1 immunoreactivity in the NAc, normalized to the mean saline intensity for each time point. (G) Quantification of microglia cell body size (μm2) measured by Iba1 immunoreactivity and normalized to the mean saline value for each time point (n = cells from four animals). (H) Representative examples of microglia processes, after 5 days of cocaine or saline. (I) Total length of microglia processes is decreased after 5 days’ cocaine by 20%, but is not significantly altered after withdrawal (n = microglia from four animals). Results are expressed as mean ± SEM, n (experiments, mice or microglia) is given in bars. ∗p < 0.05, ∗∗p < 0.01. In other brain regions, TNF-α that regulates synaptic function is produced by glia (). To assess the source of TNF-α regulating sensitization, we utilized a Cre-loxP system to selectively delete TNF-α from microglia (CX3CR1-Cre; Figure S2 A) and astrocytes (GFAP-Cre; Figure S2 B). Mice that lack microglial TNF-α showed significantly higher sensitization to cocaine from the second cocaine injection that was maintained through the period of abstinence ( Figure 2 A). Conversely, mice that lack astrocytic TNF-α did not display a significant change in sensitization compared with littermate controls ( Figure 2 B). These results suggest that microglia are important for the adaptive TNF-α response to repeated cocaine administration. To verify this, we isolated microglia from the striatum of cocaine and saline treated animals by magnetic bead sorting ( Figure S2 C) and compared TNF-α mRNA in the microglial and nonmicroglial fractions. Microglia contained the vast majority of TNF-α mRNA, showing over a 20-fold enrichment compared with the other striatal cell types ( Figure 2 C). Further, the TNF-α mRNA was increased by cocaine treatment specifically in microglia cells ( Figure 2 D) and not in other cell types ( Figure S2 D).

Kettenmann et al., 2011 Kettenmann H.

Hanisch U.K.

Noda M.

Verkhratsky A. Physiology of microglia. Resting microglia continuously survey the healthy brain and respond to a variety of activation signals by undergoing progressive morphological and functional changes (). Using Iba1, we labeled microglia in adult mice 24 hr after a single cocaine injection, 24 hr after 5 days of daily cocaine injections, or after 10 days of drug abstinence. Although the number of microglia in the NAc did not change at any time point ( Figure S2 E), Iba1 intensity was increased in microglia by 5 days of cocaine, and after a period of abstinence ( Figures 2 E and 2F). Microglia cell body size was increased by 5 days of cocaine ( Figure 2 G), accompanied by a decrease in process length ( Figures 2 H and 2I). These changes in microglia morphology are consistent with an activated phenotype. In contrast, we did not observe any activation of astrocytes, as judged by the area or intensity of GFAP expression ( Figures S2 F and S2G). These data strongly suggest that microglia are the source of the cocaine-induced upregulation of TNF-α production in the striatum observed during sensitization.

Pascoli et al., 2012 Pascoli V.

Turiault M.

Lüscher C. Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour. Casella and Mitchell, 2008 Casella C.R.

Mitchell T.C. Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant. Michaud et al., 2013 Michaud J.P.

Hallé M.

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et al. Toll-like receptor 4 stimulation with the detoxified ligand monophosphoryl lipid A improves Alzheimer’s disease-related pathology. Pascoli et al., 2012 Pascoli V.

Turiault M.

Lüscher C. Reversal of cocaine-evoked synaptic potentiation resets drug-induced adaptive behaviour. Figure 4 MPLA Activates Microglia in the Nucleus Accumbens and Decreases Behavioral Sensitization to Cocaine via TNF-α Show full caption (A) Representative confocal projection images of Iba1 immunostaining in the NAc, after MPLA (10 μg) or saline injection in mice after 10 days’ withdrawal. Scale bar, 20 μm. Semiquantification of immunoreactivity reveals that Iba1 intensity was significantly increased 24 hr after a single MPLA injection. (B) MPLA (10 μg) significantly increases TNF-α mRNA in the ventral striatum at 4 hr and 24 hr. (C) Representative traces and AMPA/NMDA ratios from D1-MSNs in the NAc core 24 hr after MPLA (10 μg) or saline injection in mice after 10 days’ withdrawal. (D) MPLA does not reduce the initial locomotor response to cocaine. Mice were given seven daily saline injections, then after 9 days of abstinence given an injection of saline or MPLA (10 μg), followed the next day by a challenge dose of cocaine. MPLA did not alter the response to the challenge dose. This response was lower than the sensitized response in control animals given cocaine during training (n = 8 sal/sal, 7 sal/MPLA, 6 coc/sal). (E) MPLA did reduce the sensitized response to cocaine. After withdrawal, WT mice were injected with MPLA (10 μg or 50 μg) or saline and tested 24 hr later with a challenge dose of cocaine (n = 20 for control, 12 for 10 μg MPLA, and 10 for 50 μg MPLA). (F) MPLA treatment had no effect on sensitization in TNF-α−/− mice, as MPLA (10 μg) did not reduce the response to a challenge dose of cocaine in TNF-α−/− mice (n = 10 saline, 11 MPLA). Results are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Our data suggest that the activation of microglia limits the cocaine-induced changes to NAc circuitry, but this activation occurs only during a narrow window following cocaine exposure. Because depotentiation of MSNs reduces cocaine-induced behavioral sensitization (), we tested if reactivation of microglia to increase TNF-α could depress NAc synapses and suppress sensitization. To do this, we utilized monophosphoryl lipid A (MPLA), a detoxified variant of LPS (). MPLA is a weak TLR4 agonist that does not induce extensive neuroinflammation or sickness behavior (). We first verified that MPLA activates microglia in the NAc, by injecting 10 μg MPLA IP after 10 days of abstinence from cocaine. MPLA treatment significantly increased Iba1 intensity within 4 hr compared to saline treated controls ( Figure 4 A) and was associated with an increase in striatal TNF-α expression, at both 4 and 24 hr after injection ( Figure 4 B). We next tested if MPLA would depress synaptic strength in the NAc core. After 10 days of abstinence from cocaine, mice were injected with MPLA and evaluated 24 hr later for AMPA/NMDA ratios on D1-MSNs. MPLA treatment significantly reduced AMPA/NMDA ratio in D1-MSNs compared to saline-treated controls ( Figure 4 C). Because artificially reducing synaptic strength in the NAc can reduce behavioral sensitization (), these data suggest that MPLA might suppress drug-induced behaviors.