Identification of immune targets altered by cocaine

To identify potential soluble factors in blood associated with cocaine use, serum from mice treated with 10 daily doses of cocaine (Fig. 1a, b—20 mg kg−1 i.p.) or 10 days of self-administration (Fig. 1c, d—0.5 mg kg−1 for each infusion) was processed 24 h after the final dose for multiplex analysis of 32 cytokines, chemokines and growth factors. The values for each analyte, represented as the fold-change from the respective saline group, are shown as a heatmap in Fig. 1e (Experimenter admin: n = 11 saline, 15 cocaine; Self-admin: n = 6 saline, 10 cocaine; *p < 0.05; **p < 0.01). Raw pg/ml values for each analyte and exact p values are available in Supplementary Data 1. To identify potential targets for further study, several factors were considered. First, if the effects were due directly to cocaine exposure, we expected the analyte to be significantly altered in the same direction in both experimenter and self-administered cocaine paradigms. Second, if it was related to behavioral response to cocaine, we expected the analyte to be correlated with cocaine sensitization and/or intake during the self-administration period. Several analytes were significantly affected by cocaine exposure, but only two–G-CSF (Experimenter-Admin: two-tailed Student’s t-test; t (24) = 2.48, p = 0.020; Self-Admin: t (9.4) = 2.51, p = 0.032) & interleukin-1α (IL-1α; Experimenter-Admin: t (13.6) = 2.19, p = 0.047; Self-Admin: t (13) = 7.29, p < 0.0001)—showed a statistically significant change in the same direction in both paradigms (Fig. 1e). Interestingly, while some analytes that were significantly regulated in only one paradigm showed a trend in the same direction in the other paradigm (e.g., KC, MIP-2), there were several that showed a relatively strong regulation in one cocaine administration paradigm, but no change at all in the other (for statistics and values, please see Supplementary Data 1). The biological significance of these discrepancies is not clear from these experiments, but our findings highlight important differences between the two administration paradigms.

Fig. 1 Serum multiplex analysis after self- and experimenter-administered cocaine in mice. a Timeline of experimenter-administered chronic cocaine injections. b Cocaine resulted in robust locomotor sensitization (n = 10 saline, 9 cocaine; two-way ANOVA–time: F (1,17) = 7.795, p = 0.013; cocaine treatment: F (1,17) = 326.5, p < 0.0001; interaction: F (1,17) = 9.035, p = 0.0080; p < 0.001 vs control). c Timeline of cocaine self-administration in mice (saline: n = 6 saline; cocaine: n = 10). d Average daily intake of cocaine across self-administration sessions. e Multiplex serum analysis of 32 chemokines, cytokines, and growth factors after experimenter- or self-administered cocaine. For each analyte, the heatmap depicts fold-change values compared to the respective saline group. Raw pg/ml values for each analyte and exact p values are available in Supplementary Data 1. f Correlation heatmap of individual analyte levels with either locomotor sensitization (Day 10/Day 1) or daily intake of cocaine. Exact r values for each analyte and exact p values are available in Supplementary Data 2. g G-CSF is increased after both experimenter- (two-tailed Student’s t-test; t (24) = 2.48, p = 0.020) and self-administered cocaine (t (9.40) = 2.51, p = 0.032), and G-CSF levels correlate with both h locomotor sensitization (Pearson’s r = 0.771, p = 0.025) and i daily intake of self-administered cocaine (r = 0.768, p = 0.026). j MIG is increased only after self-administered cocaine (t (10.3) = 3.74, p = 0.0036), and k, l individual MIG levels correlate only self-administered cocaine (r = 0.766, p = 0.027). m Levels of IL-1α are decreased after both experimenter-delivered (t (13.6) = 2.19, p = 0.047) and cocaine self-administration (t (13) = 7.29, p < 0.0001), however n, o IL-1α levels did not correlate with either behavior. Data represented as mean ± s.e.m. (*p < 0.05, **p < 0.01, ***p < 0.001 for Holm–Sidak post-hoc tests and t-tests) Full size image

As a marker of which differentially expressed analytes might be playing a role in behavioral responses to cocaine, we performed a correlation analysis between the serum level of each analyte with level of sensitization (in the i.p. injection paradigm), and the amount of average daily cocaine intake (in the self-administration paradigm). A heatmap of Pearson’s r values for all analytes is presented in Fig. 1f (full correlation matrices with exact r and p values are available in Supplementary Data 2. Interestingly, only serum levels of G-CSF were increased by both experimenter-delivery (two-tailed Student’s t-test t (24) = 2.48, p = 0.020) and cocaine self-administration paradigms (Fig. 1g; t (9.40) = 2.51, p = 0.032) and showed positive linear correlation with extent of both sensitization (Fig. 1h; Pearson’s r = 0.771, p = 0.025) and self-administration (Fig. 1i; r = 0.768, p = 0.026). While G-CSF showed both regulation and correlation in both paradigms, this was not true for all regulated cytokines. Monokine-induced by gamma interferon (MIG, also known as CXCL9) was upregulated only after cocaine self-administration (Fig. 1j; t (10.3) = 3.74, p = 0.0036), and showed positive correlation with levels of self-administration (Fig. 1l; r = 0.766, p = 0.027). However, levels of MIG were completely unaffected by experimenter-administered cocaine (Fig. 1j; t (24) = 0.177, p = 0.86) and unrelated to cocaine-induced sensitization (Fig. 1k; r = −0.361, p = 0.38). Similarly, levels of IL-1α were significantly downregulated in both cocaine administration paradigms (Fig. 1m—exper-admin: t (13.6) = 2.19, p = 0.047; self-admin.: t (13) = 7.29, p < 0.0001), but showed no correlation with either behavior (Fig. 1n, o). Based on this evidence for the unique regulation of G-CSF by cocaine, it was chosen as the subject of more in depth functional analyses.

Identifying the effects of G-CSF on neuronal activation

Because we found that serum G-CSF levels were highly correlated with the extent of cocaine experience, we aimed to define where in the brain it might influence cocaine responses. Despite being a ~20 kDa protein, G-CSF is a soluble factor that readily crosses the blood brain barrier15,16. To identify G-CSF-responsive brain regions, we assessed transcript levels of the immediate early gene c-Fos, which is used as a marker of neuronal activation17, after an acute i.p. injection of cocaine across several regions implicated in the motivation to self-administer cocaine: medial prefrontal cortex (mPFC), nucleus accumbens (NAc), dorsal striatum, ventral hippocampus, and basolateral amygdala (Fig. 2a). In the medial prefrontal cortex we saw the expected main effect of cocaine (Fig. 2b; two-way ANOVA F (1,27) = 16.41, p = 0.0004), a main effect of G-CSF (F (1,27) = 5.243, p = 0.030), and a trend towards a cocaine by G-CSF interaction (F (1,27) = 3.759, p = 0.063). Post-hoc analysis revealed significantly enhanced c-Fos induction when comparing cocaine alone to cocaine + G-CSF (Holm–Sidak post-hoc: p = 0.026), but no effect when comparing saline to saline + G-CSF (p = 0.80). We saw a similar pattern in the nucleus accumbens with a main effect of cocaine (Fig. 2c; F (1,29) = 33.62, p < 0.0001), as well as a statistically significant main effect of G-CSF (F (1,29) = 6.803, p = 0.014) which was driven by a G-CSF by cocaine interaction (F (1,29) = 5.215, p = 0.030). As with the medial prefrontal cortex, in the nucleus accumbens of mice pre-treated with G-CSF there was significantly enhanced potentiation of c-Fos expression after cocaine compared to cocaine alone (p = 0.0059). This pattern was not seen in the other brain regions analyzed: there was a significant main effect of cocaine increasing c-Fos levels in the dorsal striatum (Fig. 2d; F (1,28) = 20.76, p < 0.0001) and the ventral hippocampus (Fig. 2e; F (1,24) = 11.46, p = 0.0024), but there were no main effects of G-CSF or any significant interactions, and in the basolateral amygdala we found no significant main effects or interactions (Fig. 2f).

Fig. 2 G-CSF potentiates cocaine-induced neuronal activation in specific brain regions. a Experimental Timeline (left). Mice were i.p. injected with G-CSF (50 µg kg−1) or PBS 24 h and again 30 min before an injection of cocaine (20 mg kg−1 i.p.) or saline and brain tissue was collected 90 min later. c-Fos expression was measured in critical brain regions involved in the motivation to self-administer cocaine (right): G-CSF enhanced cocaine-induced neuronal activation in the mPFC and NAc. b mPFC (two-way ANOVA – cocaine: F (1,27) = 16.41, p = 0.0004; G-CSF: F (1,27) = 5.243, p = 0.030; interaction: F (1,27) = 3.759, p = 0.063), c NAc (cocaine: F (1,29) = 33.62, p < 0.0001; G-CSF: F (1,29) = 6.803, p = 0.014; interaction: F (1,29) = 5.215, p = 0.030) d While cocaine increased neuronal activation in the dorsal striatum (two-way ANOVA –cocaine: F (1,28) = 20.76, p < 0.0001), there was no added effect of G-CSF (F (1,28) = 0.05115, p = 0.82) e Similar results were observed in the ventral hippocampus (F (1,24) = 11.46, p = 0.0024; G-CSF: F (1,24) = 0.07447, p = 0.79). f c-Fos was not significantly induced by cocaine in the basolateral amygdala (F (1,31) = 2.463, p = 0.13). g c-Fos expression levels were correlated between the NAc and mPFC (Pearson’s r = 0.904, p < 0.0001). Data represented as mean ± s.e.m. (*p < 0.05, **p < 0.01, ***p < 0.001 for Holm-Sidak post-hoc tests) Full size image

There are extensive projections from the mPFC to the NAc—the only two regions that showed a significant interaction between G-CSF and cocaine—and further, the levels of c-Fos expression between these two regions was found to be correlated (Fig. 2g; Pearson’s r = 0.904, p < 0.0001), suggesting the possibility that mPFC to NAc projections, already strongly implicated in behavioral effects of cocaine18, may be playing a critical role in this process, possibly via glutamatergic projections driving further activation in the NAc.

Regulation of G-CSF gene expression in the NAc and mPFC

Given that G-CSF treatment controls patterns of neuronal activation in the NAc and mPFC in response to cocaine, we examined how treatment with cocaine might affect the expression of G-CSF and its receptor locally within these regions. Transcript for G-CSF itself (Csf3) was significantly induced in both regions 90 min after an acute (20 mg kg−1) i.p. injection of cocaine (Fig. 3a; two-tailed Student’s t-test—NAc: t (17) = 2.60, *p = 0.019; mPFC: t (8) = 3.06, p = 0.016). In contrast, when animals were analyzed 24 h after a seven-day course of cocaine (20 mg kg−1 per day, i.p.), levels of Csf3 had increased even further in the NAc (Fig. 3b; NAc: t (42) = 3.57, p = 0.0009), but were not significantly changed in the mPFC (Fig. 3b; t (27) = 1.15, p = 0.26). Similarly, after this prolonged treatment with cocaine we found that levels of the G-CSF receptor were also increased in the NAc (Fig. 3c; t (24.1) = 2.71, p = 0.012) but not in the mPFC (Fig. 3c; t (20) = 0.853, p = 0.40). It is unclear as to why there were not changes in the mPFC, however, it is possible that changes in signaling from the receptor, not just relative expression levels could be playing a role. These findings, in conjunction with the c-Fos data, suggest strongly that the NAc is a crucial region for G-CSF signaling in response to cocaine.

Fig. 3 G-CSF-related gene expression is increased after cocaine. a, b mRNA levels of Csf3 (G-CSF) in the NAc and mPFC after acute (a—two-tailed Student’s t-test – NAc: t (17) = 2.60, p = 0.019; mPFC: t (8) = 3.06, p = 0.016) and 7 days of i.p. cocaine injections (b, NAc: t (42) = 3.57, p = 0.0009; mPFC: t (27) = 1.15, p = 0.26). c mRNA levels of Csf3r (G-CSF receptor) after 7 days of i.p. cocaine injections in the NAc (t (24.1) = 2.71, p = 0.012) and mPFC (t (20) = 0.853, p = 0.40). Data represented as mean ± s.e.m. (*p < 0.05, ***p < 0.001 for t-tests) Full size image

Previous studies have shown that G-CSF and its receptor are expressed in forebrain neurons16,19 and multiple subtypes of glial cells20,21, however, the expression pattern in the NAc remains unknown. The primary output neurons in the NAc are composed of two primarily non-overlapping populations of GABAergic medium spiny neurons (MSNs), defined by the predominant expression of D1 or D2 dopamine receptors. Thus, we performed immunohistochemistry to examine both expression levels of G-CSF and its receptor in these two subpopulations using mice that express tdTomato under the control of the D1 receptor promoter, and thereby identifying both D1+ and D1− populations. G-CSFR was highly expressed in both D1+ and D1− cell populations (Fig. 4a upper), suggesting expression in both cell types. In addition to expression in cells that morphologically resemble MSNs, expression was detected in surrounding glial cells.

Fig. 4 Detection of G-CSF and G-CSFR in the NAc of D1-tdTomato mice. Immunolabeling for tdTomato protein and GCSFR or GCSF in D1-tdTomato mice was performed to determine cell-type expression in the NAc. a Representative confocal images acquired in the shell of the NAc from control animals, demonstrating expression of G-CSFR (upper) and G-CSF (lower) in multiple cell types. b Representative images from mice treated with cocaine (20 mg kg−1, i.p. × 7 days) again showing expression of G-CSFR (upper) and G-CSF (lower). For all images nuclei were counterstained with DAPI, tdTomato protein is labeled in red, G-CSF and G-CSFR are labeled in green. Scale bar = 20 µm Full size image

Similarly, G-CSF itself seems to be expressed in a diverse array of cell types (Fig. 4a lower), and while there is clear peri-neuronal staining surrounding D1+ MSNs, there was no specific cell pattern that emerged from the staining. Interestingly, when these same transgenic mice were treated with 7 days of experimenter-administered cocaine, there was no apparent shift in cell expression pattern of either G-CSF or its receptor (Fig. 4b). These results are consistent with findings from the periphery where G-CSF is expressed in myriad cell types including monocytes, endothelial cells, and fibroblasts22,23,24, and suggest the possibility that the effect of G-CSF on behavior is occurring through effects on multiple cell types.

G-CSF in the NAc is increased by mPFC to NAc stimulation

While there were clear increases in G-CSF expression in NAc following cocaine exposure, we aimed to elucidate the underlying mechanisms. To this end, a two-part experiment was designed to determine if central or peripheral actions of cocaine were critical in G-CSF induction. First, we aimed to determine if increases in the activity of specific projection pathways in brain were capable of increasing central or peripheral G-CSF levels using designer receptors exclusively activated by designer drugs (DREADDs). Second, using a cocaine analog (cocaine methiodide) that does not cross the blood-brain barrier, we tested the effects of increased peripheral cocaine on central and peripheral G-CSF levels.

In the first series of experiments, a retrograde traveling CAV2 virus that drove the expression of Cre-recombinase was injected into the NAc. Cre-dependent expression of the excitatory G q -coupled DREADD receptor was then induced in either the mPFC or the ventral tegmental area (VTA). This allows for pathway-specific stimulation of the mPFC to NAc or VTA to NAc projections (Fig. 5a). 7 days of daily clozapine-N-oxide (CNO) injections (1 mg kg−1, i.p.) robustly increased the expression of Csf3 in the NAc after mPFC to NAc, but not after VTA to NAc, stimulation (Fig. 5b One-way ANOVA - Main effect: F (2,12) = 13.4, p = 0.0009; Sidak post-hoc: p = 0.0037). A similar effect was found for expression of the G-CSF receptor gene Csf3r (Fig. 5c; Main effect: F (2,12) = 8.14, p = 0.0058; Sidak post-hoc: p = 0.0093). Stimulation of these two pathways did not have any significant effect on serum levels of G-CSF (Fig. 5d; Main effect F (2,12) = 1.82, p = 0.20).

Fig. 5 G-CSF levels are increased by the selective activation of mPFC to NAc projections. a Experimental design of projection-specific DREADD stimulation. Mice were injected with a retrograde traveling CAV2-Cre virus in the NAc and a Cre-dependent hM3Dq-DREADD virus in either the mPFC or the VTA to allow for the specific stimulation of either mPFC to NAc or VTA to NAc. b Csf3 (G-CSF) mRNA levels in the NAc were increased after mPFC to NAc stimulation (one-way ANOVA; F (2,12) = 13.4, p = 0.0009, Sidak post-hoc: p = 0.0037 vs control). c Csf3r (G-CSFR) mRNA levels in the NAc were increased only after mPFC to NAc stimulation (F (2,12) = 8.14, p = 0.0058, p = 0.0093 vs control). d Peripheral G-CSF serum levels were not affected by stimulation (F (2,12) = 1.82, p = 0.20). e Mice were injected i.p. for 7 days with cocaine methiodide (CocMet), a cocaine analog that does not cross the blood brain barrier, to assess the effects of peripheral cocaine on G-CSF. Cocaine methiodide chronic treatment had no effect on f Csf3 (G-CSF) mRNA levels in the NAc (two-tailed Student’s t-test; t (12) = 0.772, p = 0.45), g Csf3r (G-CSFR) mRNA levels in the NAc (t (12) = 1.11, p = 0.29), or h G-CSF serum levels (t (12) = 0.631, p = 0.54). Data represented as mean ± s.e.m. (**p < 0.01 for Sidak post-hoc tests) Full size image

Next, we assessed whether the peripheral effects of cocaine, independent of any centrally-mediated actions, were sufficient to increase levels of G-CSF either in the serum or in the brain. To this end, mice were injected daily with cocaine methiodide–a charged analog of cocaine that does not cross the blood-brain barrier25 (Fig. 5e). After 7 days of treatment, we found that cocaine methiodide did not affect transcript levels of Csf3 (Fig. 5f; two-tailed Student’s t-test—t (12) = 0.772, p = 0.45) or Csf3r (Fig. 5g; t (12) = 1.11, p = 0.29) in the NAc. Interestingly, serum levels of G-CSF in cocaine methiodide-treated mice were also unchanged (Fig. 5h; t (12) = 0.631, p = 0.54). Taken together, these experiments suggest that G-CSF signaling in the NAc is regulated by specific input pathways, and that peripheral upregulation of G-CSF by cocaine likely involves feedback signaling from the CNS.

G-CSF enhances cocaine-induced locomotor sensitization

To examine a possible causal link between systemic G-CSF and locomotor sensitization to cocaine, mice were injected i.p. with PBS or G-CSF (50 μg kg−1) on the morning of each monitoring day one hour before testing (Fig. 6a). For the first 2 days, animals then received a saline injection and activity was monitored. Importantly, G-CSF on its own had no effect on locomotor behavior. Following repeated injections of cocaine, there were significant main effects of time (F (6,42) = 33.16, p < 0.0001; n = 4 PBS, 5 G-CSF) and treatment with G-CSF (F (1,7) = 8.808, p = 0.021), as well as a significant time×treatment interaction (F (6,42) = 3.942; p = 0.0032). Holm–Sidak’s post-hoc testing revealed that there were significantly different effects on days 2–5 of cocaine treatment (Fig. 6a), but not on the first day of cocaine—demonstrating that systemic G-CSF enhances locomotor responses to repeated injections of cocaine. It is noteworthy that in this set of experiments the PBS-treated animals seemed to show a delayed sensitization curve, however, the G-CSF treated animals were indeed increased when both groups seem to have reached a plateau at day 5. Importantly, given that G-CSF can mobilize immune cells from the bone marrow, we ensured that prolonged treatment with G-CSF at this dose does not cause infiltration of peripheral monocytes into the brain parenchyma (Supplementary Fig. 1).

Fig. 6 G-CSF enhances cocaine-induced locomotor sensitization and CPP. (a—top) Experimental timeline of locomotor sensitization to cocaine. Mice (n = 4 PBS, 5 G-CSF) were i.p. injected with G-CSF (50 µg kg−1) or PBS 1 h before monitoring locomotor activity following an injection of saline or cocaine (7.5 mg kg−1). (a—bottom) Locomotor sensitization to cocaine was increased in mice pre-treated with G-CSF (repeated-measures two-way ANOVA; time: F (6,42) = 33.16, p < 0.0001; G-CSF: F (1,7) = 8.808, p = 0.021; interaction: F (6,42) = 3.942; p = 0.0032). b For cocaine conditioned place preference, mice were injected with G-CSF (50 µg kg−1) or PBS 1 h every day before testing. Two-way ANOVA testing demonstrated a main effect of G-CSF (F (1,36) = 11.76, p = 0.0015), and Holm-Sidak post-hoc testing demonstrated increased CPP in G-CSF-treated mice conditioned with 3.75 mg kg−1 of cocaine (PBS n = 6; G-CSF: n = 9; p < 0.05 vs PBS), 7.5 mg kg−1 (PBS: n = 6; G-CSF: n = 10; p < 0.05 vs PBS) of cocaine but not with 15 mg kg−1 (PBS: n = 5; G-CSF: n = 6). (*p < 0.05, **p < 0.01 for Holm-Sidak post-hoc tests; # p < 0.05, ## p < 0.01 for two-way ANOVA main effects) Full size image

G-CSF alters preference formation specifically for cocaine

While locomotor sensitization reflects behavioral plasticity associated with repeated cocaine exposure, it is dissociable from the subjective “rewarding” effects of cocaine. To assess whether systemic G-CSF also alters the ability of an animal to associate predictive contextual cues with the rewarding properties of cocaine, we used an unbiased conditioned place preference (CPP) assay in mice (typically used as a measure of cocaine “reward”)26. We tested a range of cocaine doses beginning with a dose that does not lead to formation of preference in control animals (3.75 mg kg−1) up to a relatively high dose (15 mg kg−1) which does. For these experiments, animals were injected with G-CSF on each morning of the paradigm to maintain a high serum level of G-CSF. Treatment with G-CSF resulted in a significant leftward shift of the dose response curve (Fig. 6b; two-way ANOVA main effect of G-CSF: F (1,36) = 11.76, p = 0.0015) leading to the formation of a robust preference for the lowest cocaine dose–which did not result in significant preference in control animals (Holm-Sidak post-Hoc: p = 0.028; n = 6 PBS, 9 G-CSF) – and a significantly enhanced preference for the 7.5 mg kg−1 dose (Holm–Sidak post-Hoc: p = 0.035; n = 6 PBS, 10 G-CSF), but no change at the highest dose (Holm–Sidak post-Hoc: p = 0.38; n = 5 saline, 6 G-CSF).

We next determined whether G-CSF has rewarding value on its own. In the first experiment, mice were injected with G-CSF each morning similar to the treatments used in Supplementary Fig. 2a, but in this case both chambers were paired with saline. Animals did not form any preference or aversion for either chamber (two-tailed Student’s t-test: t (7) = 0.0862, p = 0.93). In addition, we determined if animals would form associations between predictive contextual cues and G-CSF itself. We found no preference or aversion that resulted from G-CSF pairings (Supplementary Fig. 2b t (8) = 0.125, p = 0.90).

We next determined if G-CSF’s modulatory effects on cocaine reward learning were specific to cocaine or generalized to natural reward-related behaviors. We thus performed a two-bottle sucrose preference task in which animals choose between a water bottle and another bottle with sucrose. Mice were treated daily with G-CSF. We found no difference between the two treatment groups (Supplementary Fig. 3a; two-tailed Student’s t-test: t (7) = 0.348, p = 0.74). Taken together, these results suggest that G-CSF enhances associative learning for drug rewards, but is not inherently rewarding or aversive, and does not affect preference for a natural reward.

Causally linking G-CSF signaling to reward learning

To determine whether G-CSF is also necessary for cocaine reward processing, we performed an experiment in which mice were injected i.p. with a G-CSF neutralizing antibody on each day of the CPP assay to reduce serum levels of G-CSF. Given that G-CSF crosses the blood brain barrier15,16, but peripherally injected antibodies do not27, this approach was intended to provide a readout of the effect of circulating G-CSF on the formation of place preference. There was a strong trend toward decreased preference in these animals, but there was no statistically significant between-group difference (Fig. 7a; two-tailed Student’s t-test: t (13) = 1.48, p = 0.16). However, given that cocaine treatment bolsters G-CSF expression in the NAc (Fig. 3), we wanted to test the possibility that the signal needed to be neutralized more proximally to the site of action. For these experiments, we infused the same G-CSF neutralizing antibody or pre-immune IgG into the NAc using osmotic mini-pumps. When we tested place preference in these animals at the same dose, we saw that blockade of G-CSF signaling in this brain region with neutralizing antibody resulted in a significant reduction in the formation of cocaine place preference (Fig. 7b; t (12.4) = 3.75, p = 0.0026).

Fig. 7 Neutralization of central G-CSF signaling reduces conditioned place preference. a To determine the role of circulating G-CSF in behavior, mice (IgG: n = 6; α-G-CSF antibody: n = 9) were i.p. injected with anti-G-CSF antibody (10 µg) or pre-immune IgG control antibody 1 h every day before testing for CPP at 7.5 mg kg−1 of cocaine. Systemic anti-G-CSF antibody did not significantly affect cocaine CPP (Two-tailed Student’s t-test: t (13) = 1.48, p = 0.16). b To test the effects of blocking signaling in the NAc anti-G-CSF antibody or pre-immune IgG (1 µg/side) was infused into the NAc via continuous osmotic minipump before and during testing for CPP at 7.5 mg kg−1 of cocaine (IgG: n = 5; α-G-CSF antibody: n = 10). NAc infusion of anti-G-CSF antibody blocked cocaine CPP (t (12.4) = 3.75, p = 0.0026). Data represented as mean ± s.e.m (**p < 0.01 for t-test) Full size image

G-CSF alters the motivation to voluntarily consume cocaine

CPP provides a measure of associative learning. Thus, changes in attention, learning mechanisms, or the rewarding properties of the stimulus can all result in increased place preference. To dissociate changes in reward, drug consumption, and motivation from one another, we used drug self-administration in rats to understand how G-CSF alters the motivational properties of cocaine. Figure 8a outlines the timeline for these experiments. Rats were trained to acquire cocaine self-administration on a fixed-ratio one (FR1, 0.8 mg kg−1 per infusion) schedule of reinforcement, and then split into two equally balanced groups with no significant differences in either acquisition (Fig. 8b) or cocaine intake (Fig. 8c) over the acquisition sessions before treatment with G-CSF or PBS, indicating that the groups were appropriately counterbalanced.

Fig. 8 G-CSF increases the motivation to self-administer cocaine. a Experimental timeline. Rats were trained to self-administer cocaine on a fixed-ratio one (FR1) schedule of reinforcement. Animals went through two behavioral tasks: the threshold procedure to assess motivation, and FR1 self-administration to assess consumption. b Acquisition for the two experimental groups before treatment showing that there were no significant differences before the experiment (two-way ANOVA: time: F (4,64) = 69.11, p < 0.0001; group: F (1,16) = 0.00147, p = 0.97; interaction: F (4,64) = 0.1985, p = 0.94). c Cocaine intake did not differ in the two groups before G-CSF or PBS treatment (Student’s t-test; t (16) = 0.427, p = 0.34). d Dose-response curves. G-CSF pretreatment increased responding for lower doses of cocaine indicating an upward shift in the dose–response function (two-way ANOVA; price: F (9,135) = 42.06, p < 0.0001; G-CSF: F (1,15) = 4.623, p < 0.05; interaction: F (9,135) = 2.616, p < 0.01). e, f Representative demand curves, plotting consumption of cocaine as a function of price, from a PBS-treated control and G-CSF-treated animal. g Averaged demand curves from both groups (two-way ANOVA; price: F (9,135) = 72.88, p < 0.0001; G-CSF: F (1,15) = 5.036, p = 0.40; interaction: F (9,135) = 0.9677, p = 0.47) h P max is increased in animals treated with G-CSF (Student’s t-test; t (14) = 2.002, *p = 0.033). i Q 0 levels are increased in G-CSF treated animals (t (14) = 2.374, *p = 0.016). j Intake was also measured using a fixed ratio one schedule of reinforcement. Animals treated with G-CSF took more cocaine injections in a three-hour session than their PBS-treated counterparts (Student’s t-test for active lever presses; t = 2.866, df = 15, p = 0.012). Data represented as mean ± s.e.m (*p < 0.05, **p < 0.01, p < 0.001 for Holm–Sidak post-hoc tests and t-tests; # p < 0.05 for two-way ANOVA main effects) Full size image

Rats were then treated with G-CSF (50 μg kg−1 i.p.) on the evening following the last acquisition session and each morning 30 min before each self-administration testing session to ensure that G-CSF levels were adequately increased during testing. First, animals underwent testing using a behavioral economics threshold procedure, which is a within-session method used to assess an animal’s motivation to self-administer a reinforcer in the face of increasing price (in this model price is equated to the number of responses that the animal must emit to obtain 1 mg of drug)28,29. Figure 8d shows the dose-response curve indicating that G-CSF-treated animals lever-pressed more at lower doses as compared to PBS-treated animals. It is important to note that in this experiment price is inversely related to dose, where an increase in price (in responses per mg) is because the dose is low. Animals pre-treated with G-CSF increased the number of responses for lower doses of cocaine (Fig. 8d; repeated-measures two-way ANOVA: main effect of G-CSF: F (1,15) = 4.623, p = 0.048).

The threshold procedure is particularly powerful because it allows for the application of economic principles to drug consumption28. In this task, animals self-administer cocaine on an FR1 schedule of reinforcement with no time outs. The dose starts high, therefore the price the animal has to pay in effort (relative price = number of responses that the animal must emit to obtain 1 mg of drug) is very low. As the session progresses, the dose is lowered, and animals’ response rates will increase. While the responses increase, the relative drug consumption stays the same. This allows the data to be plotted as demand curves by plotting consumption as a function of price. Animals will maintain a preferred drug level when the cost is low and continue maintaining this level as the price of cocaine increases. Eventually the cost becomes too great and the animal will choose to reduce its intake. The inflection point of the curve occurs at P max , the maximal price the animal is willing to pay for its preferred dose of drug. Animals pretreated with PBS had a P max of ~150 responses per milligram (Fig. 8e). Animals pretreated with G-CSF had significantly higher P max at around 225 responses per milligram (Fig. 8f, h; two-tailed Student’s t-test: t (14) = 2.002, p = 0.033). Group demand curves were significantly different (Fig. 8g; repeated-measures two-way ANOVA: main effect of price: F (9,135) = 72.88, p < 0.0001; main effect of G-CSF: F (1,15) = 5.036, p = 0.04), further substantiating the interpretation of an increase in motivation induced by G-CSF.