Chronic pain is a significant contributor to the worldwide burden of disease, affecting up to 30% of the population in the United States.43 Epidemiological studies reveal a possible link between the presence of pain and alcohol use disorder (AUD).2 Patients with a history of AUD usually suffer from pain syndromes during withdrawal, being peripheral neuropathy one of the most common.10,12 Conversely, there is also evidence that the presence of pain may alter alcohol consumption. In fact, treatment-seeking alcoholics have a highly prevalence of pain3,44 and higher levels of pain correlate with higher risk of alcohol relapse.20 This interplay between pain and addiction has been commonly reported for opioids,8,28 and it has also been thoroughly investigated in preclinical studies.
There is evidence that pain causes alterations in the mesocorticolimbic system (MCLS), a key region in affection, motivation, and drug addiction.24,26,40 In particular, inflammatory pain desensitizes mu-opioid receptors (MORs) in the MCLS,6 what reduces morphine-induced conditioned place preference (CPP)29,33 and drives to higher heroin consumption.19 Many similarities between opioids and ethanol mechanism of action have been evidenced, and it is well established that MORs play a critical role in ethanol action over the MCLS.30 Indeed, different behavioral approaches have described that blockade of local MORs in the ventral tegmental area (VTA) reduces or suppresses ethanol-induced reinforcing behaviors.7,16,23,35 Therefore, if the presence of pain induces alterations in the function of the MORs located in the MCLS, it could be possible that it also affects ethanol action over this dopaminergic pathway. As a consequence, pain may also have an effect on ethanol reinforcing properties and relapse. To this extent, some preclinical research has recently shown an increase in voluntary ethanol consumption in pain-suffering male mice when compared with pain-free groups.5,49 However, there is still a lack of preclinical data exploring the possible effect of pain on ethanol relapse-like behaviors. Relapse constitutes a major obstacle for AUD treatments; hence, 50% of treated people do not achieve remission after long follow-up periods.15 Moreover, and as previously mentioned, clinical studies have shown that the correct management of the pain situation is associated with lower risk of alcohol relapse.20 Therefore, it is worth to further investigate whether pain condition can alter alcohol relapse, and if so, which could be the contributing neural mechanisms. Although different behavior may be observed depending on the sex, based in previous data that have shown an effect in alcohol intake in males but not in females, we decided to perform our studies in male rats.49 To this end, in this study, we use an inflammatory pain model to evaluate changes in: (1) ethanol-evoked dopamine (DA) release in the nucleus accumbens (NAc); (2) context-induced associations by analyzing intra-VTA ethanol-induced place preference; and (3) ethanol-relapse by evaluating the appearance of the alcohol deprivation effect (ADE) in long-term ethanol experienced rats.
2.1. Rats housing
Male albino Wistar rats (Envigo, Barcelona, Spain; 300-340 g at the time of surgery or 280-300 g at the beginning of the long-term ethanol self-administration) were individually housed in plastic cages (48 × 38 × 21 cm3) provided with shredded aspen bedding (Teklad, Spain) and cotton enrichment (iso-BLOXTM; Teklad) and controlled humidity and temperature (22°C), a 12:12-hour light/dark cycle (on 08:00, off 20:00), and free access to food (Teklad Global Diets) and tap water. All the procedures were performed in strict accordance with the European Commission Directive 2010/63, Spanish laws (RD 53/2013) and animal protection policies. Experiments were approved by the Animal Care Committee of the University of Valencia and authorized by the Regional Government.
2.2. Surgery and inflammatory pain model
All surgeries were performed under isoflurane (1.5 minimum alveolar concentration, MAC) anaesthesia under aseptic conditions. For microdialysis experiments, rats were implanted stereotaxically (Stoelting, Kiel, WI) with bilateral vertical concentric-style microdialysis probes containing 2 mm of active membrane (Hospal AN69; molecular cutoff 60,000 Da) constructed according to Santiago and Westerink 199033 into the NAc (anteroposterior: +1.5 mm, mediolateral: ±1.6 mm and dorsoventral = −8.0 mm from bregma).34 For CPP experiments, rats were implanted bilaterally with two 28-gauge guide cannulae aimed at 1.0 mm above the posterior VTA (anteroposterior: −6.0 mm, mediolateral: ±1.8 mm, and dorsoventral: −7.9 mm). Both coordinates have been successfully used in previous studies in our laboratory.7,17,18 Cannulae were angled toward the midline at 10° from the vertical. A stainless-steel stylet (33-gauge), extending 1.0 mm beyond the tip of the guide cannula, was put in place at the time of surgery and removed at the time of testing.
We selected the complete Freund adjuvant (CFA) model of inflammatory pain. Complete Freund adjuvant (Calbiochem) was diluted in the same volume of sterile saline before its subcutaneous injection of 0.1 mL in the plantar surface of the hind paw.19 The intraplantar (Ipl) injection of saline or CFA was performed at the same time of the surgery for the microdialysis experiments, 4 days after the cannulation surgery for the CPP experiments and 2 days before the reintroduction of ethanol for the ADE experiment.
Forty-eight hours after the sterotaxical surgery, animals belonging both to the Ipl CFA (n = 9) and Ipl saline (n = 8) group were placed in Plexiglas bowls. A PE10 inlet tubing was attached to a 2.5-mL syringe (Hamilton, Spain) mounted on a syringe pump (Harvard instruments, South Natick, MA) and connected to the probes. Probes were continuously perfused with artificial cerebrospinal fluid (aCSF) comprising 0.1 mM aqueous phosphate buffer containing 147 mM NaCl, 3.0 mM KCl, 1.3 mM CaCl2, and 1.0 mM MgCl2 (pH 7.4) at a flow rate of 3.5 μL/minutes. After a minimum stabilization period of 1 hour, samples were collected every 20 minutes and extracellular DA levels were determined immediately after collection by using offline high performance liquid chromatography (HPLC) with electrochemical detection. The HPLC system consisted of a Waters 510 series pump in conjunction with an electrochemical detector (Mod. Decade, Antec, Leyden, the Netherlands). The applied potential was = 0.55 V (ISAAC cell; Antec, Leyden, the Netherlands). Dialysates were injected into a 2.1-mm RP-18 column (Phenomenex, Spain, Gemini-NX 3 µm 100 × 2.00 mm) with a 65 μL sample loop. The mobile phase consisted of a sodium acetate/acetic acid buffer (0.05 mol/L, pH= 6) containing 140 mmol/L of sodium chloride, 200 mg/L of 1-octanesulfonic acid, 100 mg/L of EDTA, and 150 mL/L of methanol. The mobile phase was pumped through the column at a flow rate of 0.06 mL/minutes. Chromatograms were analyzed and compared with standards (1.1, 2.2, 5.5, and 11 nM) run separately on each experimental day, using the AZUR 4.2 software (Datalys, France). We selected an intrasubject design where animals received 2 s.c. administrations composed by a saline injection and a 1.5 g/kg ethanol injection 100 minutes after the first saline injection. This design, although increases the complexity of the analysis, allowed us to ensure that the observed effects were derived from the pharmacological action of ethanol, and not by the injection procedure. Thus, once DA baseline level was established (defined as 3 consecutive samples with <10% variation in DA content), 2 mL of saline were subcutaneously (s.c.) administered and DA levels were analyzed every 20 minutes for 100 minutes as a control for the possible effect of the s.c. injection itself. Right after, a single s.c. ethanol dose (1.5 g/kg diluted in 2 mL of saline) was administered and DA levels in the dialysates were analyzed for 160 minutes more. The s.c. route of administration, as i.p., simulates oral alcohol blood levels because of the pharmacokinetic properties of ethanol and s.c. route of absorption.46 Moreover, produces less stress than i.p. administration since it requires a minimum manipulation of the animal.
At the end of the microdialysis experiments, rats were killed and the brains removed and rapidly frozen in dry ice; 40 μm-thick coronal slices of the NAc core were obtained using a cryostat and stained with a cresyl violet protocol to verify proper probe placement.
2.4. Ethanol conditioned place preference
The ethanol CPP paradigm has been successfully used in previous studies in our laboratory.7 The test was performed in a nonbiased 2-compartment box connected by a removal barrier with an open door in the middle. The 2 compartments differed by the wall color: black and white vertical stripes (vertical compartment) and black and white horizontal stripes (horizontal compartment). Four days after recovery from surgery and 2 days before the “pretest,” rats received a subcutaneous administration in the hind paw of 0.1 mL saline or 0.1 mL CFA. The next day, animals were exposed to the CPP box for 5 minutes to habituate them to the apparatus. The day before the conditioning, animal natural preference for the compartments was tested during 15 minutes (pretest). During “conditioning,” rats received bilateral intra-VTA infusions before placing them in the appropriate compartment. All conditioning phases consisted on 8 sessions (2 sessions/day: morning session and afternoon session) of 30 minutes distributed in 4 days. Twenty-seven animals were randomly assigned to one of the ethanol doses tested (70 or 52 nmol), and the exposure to conditioning compartments was counterbalanced in both groups. Control group animals (n = 6, no Ipl treatment) received 8 administrations of the equivalent volume of aCSF meanwhile the other groups received ethanol or aCSF on alternate sessions. After the last conditioning session, each animal was subjected to the “test” for its place preference: The rat was placed in the open door of the barrier, and the time spent in each compartment was recorded over 15 minutes. Place preference scores were calculated as test minus pretest time spent (in seconds) on the ethanol-paired compartment.
At the end of the CPP experiments, rats were killed and the brains removed and rapidly frozen in dry ice; 40 μm-thick coronal slices of the VTA were obtained using a cryostat and stained with a cresyl violet protocol to verify proper cannula placement.
2.5. Long-term ethanol self-administration
Before initiating the long-term voluntary ethanol drinking procedure, rats (n= 20) were habituated to the animal room for 2 weeks. Next, animals were given continuous access to tap water and to 5%, 10%, and 20% (vol/vol) ethanol solutions in their home cages. Every week, animals were weighted; all drinking solutions renewed and changed the positions of the 4 bottles to avoid location preferences. After 8 weeks of continuous ethanol availability, the first 2-week deprivation period was introduced. After, rats were given access to alcohol again and 3 more deprivation periods were performed in a random manner. The duration of these drinking and deprivation periods was irregular: 6 ± 2 weeks and 2 ± 1 weeks, respectively, to prevent behavioral adaptations.31 The total amount of ethanol intake (g/Kg/day) was recorded during the whole experiment by weighing the bottles. Animals were randomly assigned to one of the 2 experimental groups. The baseline drinking for each group was considered as the average of the measurements of the 3 last days before the fourth abstinence period. During this last abstinence period and 48 hours before reintroduction of the ethanol solutions, rats received a subcutaneous administration in the hind paw of 0.1 mL saline or 0.1 mL CFA. After the reintroduction of the ethanol solutions, the daily weighing routine was restored during the 3 postabstinence days to assess the ADE.
2.6. Supervision of the inflammatory pain model
With the objective to assess the level of inflammation induced by CFA injection, we measured the dorsoventral distance of the rats injected hind paw and compared it to the contralateral hind paw distance. This measurement was performed right before sacrifice in both the microdialysis and the long-term ethanol self-administration experiments.
For the CPP experiment, we tested the mechanical nociception thresholds by using the von Frey test before and after 2 and 9 days of intraplantar injections (after the pretest and test sessions). After 20 minutes of habituation to the apparatus, nociception thresholds were measured by the manual application of 5 filaments (Aesthesio) with a simplified up-down method, as described in Bonin 2014.4 Results were expressed by the mean of nociception threshold (in grams).
2.7. Statistical methods
All data sets are expressed as mean ± SEM. In all cases, homogeneity of variance was tested and the significance level was always set at P < 0.05.
2.7.1. Microdialysis experiments
DA levels in the 3 dialysate samples defining the baseline conditions (expressed as fmol in 65 μL) were averaged to calculate baseline levels in each animal. Differences in baseline levels between groups (Ipl saline and Ipl CFA-treated animals) were evaluated using the unpaired Student t test.
DA levels were transformed to percentages of baseline for each individual rat and were statistically analyzed by a mixed two-way analysis of variance (ANOVA) for repeated measures, with group (saline or CFA injection) taken as the between-subject factor and time as the within-subject factor. Significant interaction time x group was further analyzed by means of a Bonferroni correction for multiple comparisons. Significant effects of time were analyzed by one-way ANOVA with repeated measures per each group followed by Bonferroni multiple-comparisons test and postinjection values were compared with the last baseline measure.
Areas under the curve (AUC) were calculated from 0 to 100 minutes (postsaline effect) and from 100 to 200 minutes (postethanol effect) for each rat from percentage data. Areas under the curve were statistically analyzed by mixed two-way ANOVA with repeated measures with group as a between-subject factor and treatment (saline and ethanol) as a within-subject factor followed by Bonferroni corrections for multiple comparisons when interactions were found to be significant.
2.7.2. Conditioned place preference
For CPP experiments, results are expressed in preference score, calculated as time spent in ethanol paired compartment during test minus time spent in the same compartment during pretest. Preference scores were analyzed using one-way ANOVA, followed by least significant difference (LSD) test.
For the von Frey test, results are expressed as mechanical nociceptive threshold in grams. Nociceptive thresholds were statistically analyzed by mixed two-way ANOVA with repeated measures with group (Ipl CFA or Ipl saline) as a between-subject factor and time as a within-subject factor followed by Bonferroni corrections for multiple comparisons.
2.7.3. Ethanol self-administration
Data were expressed as total amount of ethanol intake (g/Kg/day). Basal and either combined and individual measures of postabstinence ethanol intake were statistically analyzed by mixed two-way ANOVA with repeated measures with group as a between-subject factor and period (basal and postabstinence) as a within-subject factor followed by Bonferroni corrections for multiple comparisons when interactions were found to be significant.
3.1. Effect of intraplantar complete Freund adjuvant on ethanol-evoked DA release in the nucleus accumbens
The objective of this experiment was to analyze whether ethanol-induced DA signaling within the NAc is affected in this inflammatory pain model. For that, 48 hours after CFA injection, extracellular DA content in the accumbal dialysates was monitored every 20 minutes. Once a stable baseline for DA was achieved (< 10% variation in 3 consecutive samples), rats received a first saline s.c. injection to discriminate the possible effect of the manipulation/injection itself on the NAc DA levels. Next, a single ethanol dose was administered (1.5 g/kg, s.c.) and DA levels were monitored until the end of the experiment for a total of 260 minutes (Fig. 1A). Baseline DA levels (mean ± SEM) were 51.8 ± 9.8 fmol/65 μL and 62.8 ± 8.4 fmol/65 μL for the Ipl saline vs Ipl CFA group, respectively. Our results indicate that Ipl CFA does not have a significant effect on basal extracellular DA levels (t test, P = 0.517 n = 8-9).
The mixed ANOVA for repeated measures detected a significant effect in the within-subject variable. Indeed, both the time (F(16,240) = 4.195, P < 0.001), in the interaction time x group (F(16,240) = 3.506, P < 0.001), but not in the effect of group (F(1,15) = 1.140, P = 0.302). The analysis of the effect in the within-subject variable time was further analyzed by means of a one-way ANOVA for repeated measures. As expected, in Ipl saline animals, 1.5 g/kg ethanol dose induced a significant increase in DA release up to 135% from 80 to 120 minutes (180-220 minutes' time points in Fig. 1B) after its administration (one-way ANOVA for repeated measures, within-subject effect of time F(16,112) = 7.631, P < 0.001; Bonferroni multiple comparisons vs the last basal time point, t = 0 minutes: p180 = 0.023, p200 = 0.017, p220 = 0.015). Interestingly, although Ipl CFA rats experienced an ethanol-induced DA release (136% from baseline), this increase was not significantly higher compared to last value of baseline (one-way ANOVA for repeated measures, within-subject effect of time F(16,128) = 3.081 P = 0.0001, Bonferroni multiple comparisons vs t = 0 minutes: P < 0.001, p20-p260 > 0.05). Indeed, ethanol-evoked DA levels in the Ipl CFA group were significantly lower than the saline group at 60, 80, and 100 minutes after 1.5-g/kg ethanol administration (Fig. 1B; two-way ANOVA for repeated measures, interaction time × group F(16,240) = 3.506, P < 0.001; Bonferroni correction for multiple comparisons, p60 = 0.048, p80 = 0.001, p100 = 0.002). Finally, it is important to mention that saline s.c. injection induced a slight increase up to 127% and 119% in DA release (at 20 minutes' time point) both in Ipl CFA and Ipl saline animals, respectively, although this increase in DA levels was not statistically significant (Ipl CFA group: p20 = 0.688, Ipl saline group: p20 = 0.896; compared to last baseline point t = 0).
To further quantify this Ipl CFA effect on the ethanol-evoked DA release, we calculated the AUC for the following 100 minutes after each administration in both Ipl CFA and Ipl saline groups (Fig. 1C). This parameter allowed us to compare the global response to ethanol on extracellular NAc DA levels. Mean AUC values were significantly different between Ipl saline and Ipl CFA groups (two-way ANOVA for repeated measures, within-subject interaction time × group F(1,15) = 6.384, P = 0.023). The ethanol-induced total effect in the Ipl saline group was significantly higher than the total effect induced by the saline injection (Bonferroni correction for multiple comparisons, P = 0.013), whereas no significant differences were found between saline and ethanol treatments in Ipl CFA animals (Bonferroni correction for multiple comparisons, P = 0.502).
Only animals that showed correct placement of the microdialysis probe in the NAc are included in the analysis. Position of the active portion of the dialysis membrane can be inspected in Figure 1D.
3.2. Effect of intraplantar complete Freund adjuvant on ethanol-induced conditioned place preference
In this experiment, we used the CPP paradigm, which procedure is summarized in Figure 2A. This behavioral paradigm was selected to assess whether the above reported Ipl CFA induced changes in ethanol-evoked DA release could be reflected also in changes on ethanol-induced context learned associations. For that, we analyzed the ability of 2 different ethanol doses directly administered into the posterior VTA to induce CPP in Ipl saline and Ipl CFA animals, by using a similar design as in our previous work.7 Our results show that causes significant alterations on ethanol-induced CPP were observed in the inflammatory pain model used (Fig. 2B, one-way ANOVA F(4,23) = 3.685, P = 0.018, power = 0.8, partial eta-squared = 0.39, calculated Cohen's f = 0.79). Concretely, Ipl saline animals that received the lowest ethanol dose (52 nmol) showed a preference score significantly higher than the control group (146 ± 26 vs −12 ± 36, LSD test, P = 0.040), whereas the Ipl CFA group did not develop a preference for the ethanol paired compartment when compared with the control group (−18 ± 49 vs −12 ± 36, LSD correction for multiple comparisons, P = 0.920). Interestingly, the higher ethanol dose used (70 nmol) induced CPP in both Ipl saline and Ipl CFA rats, shown as preference scores significantly higher than the control group (Ipl saline: 165 ± 54, P = 0.016; Ipl CFA: 140 ± 44, P = 0.024; LSD test). Moreover, the statistical analysis showed significant difference between doses only in the Ipl CFA group (CFA: P = 0.019, saline: P = 0.804).
In addition, the results of the von Frey test performed throughout the CPP experiment are represented in Figure 2C and confirmed that Ipl saline animals maintained the mechanical nociceptive threshold as the one measured in the baseline session. On the contrary, Ipl CFA animals showed a significant decrease in the mechanical nociceptive threshold that was maintained until the performance of the CPP test session (two-way ANOVA for repeated measures, within-subject interaction time × group F(2,44) = 16.299, P < 0.001; Bonferroni correction for multiple comparisons, differences between groups: pD2 < 0.001 and pD9 < 0.001, differences from baseline, Ipl saline: pD2 = 1.000 and pD9 = 1.000, Ipl CFA: pD2 < 0.001 and pD9 < 0.001).
Only animals that showed correct placement of the cannula in the VTA were included in the analysis. Position of the cannula tip can be inspected in Figure 2D.
3.3. Effect of intraplantar complete Freund adjuvant on alcohol deprivation effect
The effect of Ipl CFA on ethanol relapse was studied by selecting a self-administration paradigm in which periods of continuous access to 4 different bottles (water and 5%, 10%, and 20% [vol/vol] ethanol) were alternate with forced abstinence periods (access only to water) (Fig. 3A). During the last abstinence period and 48 hours before ethanol reintroduction, animals were injected with saline or CFA in the hind paw and ADE was analyzed by measuring the average of ethanol consumption during the 3 days after reintroduction (Fig. 3A). After the reintroduction of alcohol solutions, both Ipl CFA and Ipl saline groups showed an increase in alcohol consumption compared with basal (calculated as average of the last 3 days before the abstinence), indicating the occurrence of ADE and no significant differences were found between groups (two-way ANOVA for repeated measures, within-subject effect of time F(1,18) = 45.599, P < 0.001; interaction time × group F(1,18) = 0.536, P = 0.474). Therefore, Bonferroni correction for multiple comparisons showed significant difference between basal and postabstinence ethanol consumption for both groups (psaline < 0.001, pCFA = 0.001). We also analyzed the individual ethanol intake for each of the 3 postabstinence days (Fig. 3B), and there was an increase after ethanol reintroduction in Ipl CFA and Ipl saline groups, with no significant differences between groups (two-way ANOVA for repeated measures, within-subject effect of time F(3,54) = 15.268, P < 0.001; interaction time × group F(3,54) = 0.306, P = 0.821). Concretely, in Ipl saline animals, total ethanol intake was significantly higher in the 3 postabstinence days compared with baseline (Bonferroni correction for multiple comparisons, pday 1 = 0.001, pday 2 = 0.019, pday 3 = 0.026), whereas in the Ipl CFA group, there were only significant differences between total ethanol intake in the first postabstinence day compared with baseline (Bonferroni correction for multiple comparisons, pday 1 = 0.001, pday 2 = 0.061, pday 3 = 1.000) (Fig. 3C).
In this article, we present evidence supporting that both, ethanol-evoked neurochemical responses and dopamine-dependent behaviors, such as CPP, are altered in male Ipl CFA animals, so that higher ethanol doses are needed to elicit similar effects to those observed in male Ipl saline rats. We also show that Ipl CFA, however, did not alter the magnitude of ADE in long-term ethanol-experienced male rats.
Pain and drug addiction share common neural circuits.2,12 It has already been reported that brain pathways that mediate addiction and affection are altered by the presence of pain.8,24,28 Opioid receptors, located in the MCLS, are believed to mediate the reinforcing properties not only of opioids but also of ethanol, through regulation of DA extracellular levels within the NAc.9,47 Previous research showed that heroin-evoked DA release in the NAc is blunted in rats under inflammatory pain condition, what drives to higher heroin dose consumption.19 Similarly, in our experiment, the administration of ethanol did not provoke a significant increase in NAc DA levels in Ipl CFA animals (Figs. 1B and C). In contrast to these results, Ipl saline rats experienced a significative DA extracellular increase of 135% from baseline, as previous published data have shown.14,21 This finding further supports the fact that Ipl CFA alters the neurochemical response of the MCLS elicited by drugs. Curiously, the heroin-evoked increases of DA release described by Hipolito and colleagues in 2015 started 15 minutes after heroin administration and differences between Ipl saline and Ipl CFA groups were found 30 and 45 minutes after this administration.19 According to our results, ethanol-evoked increases and differences between groups also appear later on time. It should be also taken into account the fact that Ipl CFA rats did not return to baseline levels before ethanol administration, despite none of the measures before this point statistically differed from baseline. For that reason, comparisons of DA levels after ethanol either to baseline DA levels (at t = 0) within the Ipl CFA group or to DA levels after EtOH in the Ipl saline group may not be ideal. However, it has been previously shown that acute stressful stimuli also increase DA levels in the NAc,1 and therefore, this initial saline injection was necessary to confirm that the ethanol-evoked increase of DA levels was not an effect derived from the procedure itself. Indeed, the global effect elicited by ethanol in the Ipl saline group was significantly higher than the saline-induced total effect. On the contrary, saline- and ethanol-induced total effect were not statistically different in Ipl CFA animals (Fig. 1C), confirming that our results are derived from the drug effect. Although the specific mechanism remains to be elucidated, our microdialysis results clearly show that Ipl CFA impairs ethanol-evoked DA release in the NAc, which may have an effect on the reinforcing properties of this drug.
It is classically accepted that drug-reward–related behaviors (such as CPP)38,48 are mediated by DA transmission within the NAc. Consequently, altered neurochemical function in MCLS can be translated into abnormal changes in these behaviors. Previous studies have reported that the presence of pain suppresses the preference for opioid-associated environments.29,33,39 Again, our present results show that local administration of 52 nmol of ethanol intra-VTA is able to induce preference for the ethanol-paired compartment only in Ipl saline animals (Fig. 2B). Very interestingly, we also show that a higher ethanol dose of 70 nmol is able to reverse the impairment of ethanol place preference (Fig. 2B) observed in our inflammatory pain model, hence resulting in similar preference scores in both Ipl saline and Ipl CFA groups. Despite the apparent small size of the sample (n = 4-7/group), the observed results are reliable supported by the size of the effect (Cohen's f = 0.79) and the power (power = 0.8) obtained in the statistical analysis (see results section). In fact, the shift of the behavior observed in Ipl CFA rats depending on the intra-VTA dose of ethanol administered provides more evidence that dopamine-dependent behaviors are altered in pain-suffering animals. In fact, in our experiments, 52 and 70 nmol of ethanol-administered intra-VTA in Ipl saline animals shows a very similar behavioral output which is the induction of context-dependent associations. On the contrary, in Ipl CFA animals, the large difference observed in the behavioral output elicited by the 2 doses is showing a shift from nonactive dose (52 nmol) for these Ipl CFA animals into an active dose (70 nmol). Moreover, our Von Frey test data confirm that no changes in the nociception were observed through the CPP experimental process, ruling out the possibility of unspecific effects derived from changes in mechanical nociception. These observed phenomena are, therefore, consistent with our previous findings where higher doses of heroin were necessary both to increase DA levels in the NAc and to elicit heroin self-administration in Ipl CFA rats.19 All in all, our results show that our inflammatory pain model alters the context-learned associations elicited by the local action of ethanol on the MCLS.
Finally, we decided to study whether this previous reported alteration in ethanol-evoked neurochemical responses and DA-dependent behaviors induced by Ipl CFA could have an effect on AUD-related behaviors. Pain condition frequently elicits negative affective states driving to alterations in reward evaluation, decision making, and motivation.2,42 Alcohol use disorder is also characterized by the abnormal persistence of negative affective states during withdrawal that can promote drug seeking and relapse.11 Thereupon, and given the recent epidemiological data showing that higher levels of pain correlate with a higher risk of alcohol relapse,20 we chose an alcohol relapse behavioral approach combined with an inflammatory pain model. We selected a long-term self-administration paradigm that has been widely used for the study of alcohol relapse-like behaviors in rodents by our and other groups and that provides predicted validity.27,31,41 As expected, our results indicate that Ipl CFA alcohol-deprived male rats developed ADE after alcohol reintroduction. However, the magnitude of the ADE did not change relative to Ipl saline animals. This fact seems to be surprisingly contradictory to the previous clinical data,20,45 although these results parallel the findings of many previous studies to show that various inflammatory and neuropathic pain models have relatively weak and transient effects on operant responding maintained by other reinforcers (food, electrical brain stimulation; see Refs. 13, 22, 32, and 37). Nonetheless, it is important to consider that relapse is a complex phenomenon and animal models try to reproduce a specific aspect of this behavior. The ADE is defined as an increase in total ethanol intake that occurs during the first days after an abstinence period. It is true that numerous preclinical studies have used this paradigm to test different pharmacological strategies aimed to suppress or reduce ethanol relapse but, to the best of our knowledge, it has never been used to show an increase of the risk of relapse.27,31 Indeed, it is possible that pain does not induce an even higher ethanol intake but increases the risk of relapse. In this case, despite differences in consumption, it would be plausible to expect higher rates of relapse in Ipl CFA animals. Therefore, further behavioral studies that allow to investigate alcohol drinking behavior in the face of vulnerability to relapse are needed and would shed more light in this aspect.
It is also important to note that pain-induced changes may differently affect addictive behaviors depending on the different stages of the AUD. In fact, previous studies using an intermittent two-bottle choice paradigm show that pain induction before alcohol exposure significantly increases total intake in male mice5,49 and it may be plausible that pain increases alcohol intake during acquisition without modifying the magnitude of ADE. On the other hand, we have only tested male rats based in the previous data reported,5,49 and it could be plausible that relapse-like behavior may be expressed differentially depending on the sex. In any case, our data highlight the necessity of finding the appropriate animal model that reflects the existing clinical evidence and allows us to study the alcohol-related behavioral implication of pain-induced alterations of the MCLS.
Altogether, our results reveal that the response of the mesocorticolimbic dopaminergic pathway to alcohol is altered in this inflammatory pain model. In fact, neurochemical and behavioral approaches point to the presence of inflammatory pain as a relevant factor in the neurobiological effects of alcohol and in alcohol addiction. The above-reported changes, however, did not allow us to model the observed increase vulnerability to relapse of pain patients in the clinical set up. These data further support the impact of pain on the reinforcing mechanisms after alcohol administration and also underscore the necessity and importance of finding an appropriate animal paradigm to determine the neural mechanisms underlying the drug-related behavioral consequences derived from pain.
Conflict of interest statement
The authors have no conflicts of interest to declare.
Supplemental video content
A video abstract associated with this article can be found at http://links.lww.com/PAIN/B17.
This work was supported by Spanish Ministerio de Economia y Competitividad' MINECO PSI2016-77895-R (L.H.). The authors thank Dr Teodoro Zornoza for equipment and funding help. The authors also thank Ms Pilar Laso for grant management and personnel of the Animal Facilities (SCSIE) at the University of Valencia for their help and effort in assuring animal welfare.
Author contributions: conceptualization: L. Hipólito; methodology: Y. Campos-Jurado, A. Polache, L. Granero, and L. Hipólito; formal analysis: Y. Campos-Jurado, A. Polache, L. Granero, and L. Hipólito; investigation: Y. Campos-Jurado, J.D. Lorente, and J.L. González-Romero; writing—original draft: Y. Campos-Jurado, and L. Hipólito; resources, L. Hipólito; supervision: A. Polache, L. Granero, and L. Hipólito; writing—review and editing: Y. Campos-Jurado, A. Polache, L. Granero, and L. Hipólito.
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