The Combination of Galanin (1–15) and Escitalopram in Rats Suggests a New Strategy for Alcohol Use Disorder Comorbidity with Depression

Alcohol use disorder (AUD) is highly prevalent, and over 50% of AUD patients also suffer major depressive disorders. Selective 5-HT reuptake inhibitors (SSRIs) can reduce rodent ethanol drinking but exert modest clinical efficacy in alcoholic individuals. Finding new pharmacological strategies that could modulate alcohol consumption and depression is necessary. We have analyzed the effect of Galanin (1–15) [GAL(1–15)] on escitalopram (ESC)-mediated effect in alcohol consumption using the alcohol self-administration test, the nuclei involved in the effect, and whether GAL(1–15) + ESC modulated the response in despair or anxiety tests in animals under chronic alcohol intake. GAL(1–15) + ESC combination substantially reduced alcohol intake in the alcohol self-administration test and, moreover, enhanced the reduction of reward capacity of ESC on different reinforcers such as sucrose or saccharine. GAL(1–15) + ESC coadministration significantly decreases the number of C-Fos-IR TH cell bodies in the VTA, and PCA analysis suggests that one functional network, including VTA, RMTg and DR, is involved in these effects. Significantly in rats with chronic alcohol consumption, GAL(1–15) reversed adverse ESC-mediated effects in the depression-related behavioural test and forced swimming test. The results open up the possibility of using GAL(1–15) in combination with the SSRI Escitalopram as a novel strategy in AUD comorbidity with depression.


Introduction
Alcohol use disorder (AUD) is a highly prevalent psychiatric disorder, and over 50% of treated AUD patients also suffer from other psychiatric disorders, including major depressive disorders [1]. The rate of AUD and depression comorbidity have been assessed in epidemiologic studies that linked AUD to 3.7-fold higher odds of experiencing a depressive episode in the prior year [2], and individuals with a lifetime diagnosis of major depression to a 1.3-fold increased risk of AUD [3]. Furthermore, these studies found that major depression worsened the symptoms of AUD and vice versa [4], which may explain why people with comorbid depression and AUD are at greater risk for suicide [5,6].
Both psychiatric disorders, AUD and depression, are associated with dysregulated brain monoaminergic systems including the serotoninergic (5-HT) system. A link between serotoninergic neurotransmission and alcohol intake is suggested based on preclinical effects in alcohol consumption, we analyzed the immunohistochemistry of immediate early gene C-Fos as an indirect marker of neural activity. We studied the expression of C-Fos expression after the administration of GAL(1-15) + ESC in several nuclei involved in depression and reward-seeking behaviour-lateral (LHb) and medial (mHb) habenula, nucleus accumbens (NAc), prefrontal cortex (CPF) and the rostromedial tegmental nucleus (RMTg)-and performed double immunohistochemical staining of 5-hydroxytryptamine (5-HT) and C-Fos or tyrosine hydroxylase (TH) and C-Fos to study the specific cell activation in the dorsal raphe (DR) and ventral tegmental area (VTA), respectively. Additionally, we assessed the brain circuits using principal component analysis (PCA) in the multivariate analysis used to understand brain functional organization.

Animals
Male Sprague Dawley rats (body weight 225-250 g) were obtained from criffa and maintained in a humidity-controlled and temperature-controlled (20-22 • C) room. During the entire protocol, rats were maintained on a 12-h reversed light/dark cycle (lights off at 9 a.m.). All animal experimentation was conducted in accordance with the University of Málaga Guidelines for the Care and Use of Laboratory Animals (Ethic Code: 22/05/2017/066).
Detailed descriptions are available in the supplementary information on the animal controlled-conditions, surgical preparation and administration of substances and drugs.
In the experiment, first, a dose-response curve of ESC was performed. Groups of rats received three separate intraperitoneal injections of ESC 23, 5 and 1 h before the beginning of the tests at the doses of 7.5 mg/kg or 10 mg/kg or vehicle.
Once we determined the ESC effect, we studied the effect of the coadministration of ESC and GAL(1-15) on sucrose intake and in the sucrose preference test. For this, groups of rats received three separate intraperitoneal injections of vehicle or ESC (10 mg/kg) 23, 5 and 1 h before the beginning of the test and one intracerebroventricular (icv) injection of GAL(1-15) 1 nmol or aCSF 15 min before the test. The sucrose intake and preference were measured for 2 h, beginning 15 min after administering GAL(1-15) or aCSF.
First, a dose-response curve of ESC was calculated. Groups of rats received three separate intraperitoneal injections of ESC 23, 5 and 1 h before the tests at the doses of 2.5 mg/kg; 5 mg/kg; and 7.5 mg/kg or vehicle.
Groups of rats received three separate intraperitoneal injections of vehicle or ESC (2.5 mg/kg or 7.5 mg/kg) 23, 5 and 1 h before the beginning of the test and one icv injection of GAL(1-15) 1 nmol or aCSF 15 min before the test.
Groups of rats received three separate intraperitoneal injections of vehicle or ESC (2.5 mg/kg) 23, 5 and 1 h before the beginning of the test and one icv injection of GAL(1-15) 0.3 nmol or aCSF 15 min before the test. The number of alcohol reinforcement and number of active lever presses were assessed with the self-administration test.
In the second series of experiments, the rats with chronic alcohol consumption received three separate intraperitoneal injections of vehicle or ESC (7.5 mg/kg) 23, 5 and 1 h before the beginning of the tests and one icv injection of GAL(1-15) 1 nmol or aCSF 15 min before the depression-related tests FST and TST. The dose of ESC 7.5 mg/kg was based on our previous works in FST and TST [31].
Finally, the rats with chronic alcohol consumption received three separate intraperitoneal injections of ESC 23, 5 and 1 h before the beginning of the tests at the doses of 2.5 mg/kg or vehicle and one icv injection of GAL(1-15) 0.3 nmol or aCSF 15 min before the test to evaluate the effects in the anxiety-related tests EPM and OFT.

Behavioural Assessment (SPT)
Reward capacity was assessed using the SPT, performed as described previously [24]. Briefly, on the testing day, rats were allowed free access to two bottles: one containing 1% (w/v) sucrose solution and the other containing tap water. After 2 h, the bottles were weighed to calculate the sucrose intake (g/kg) and sucrose preference [sucrose preference = (sucrose consumption/(water + sucrose consumption) × 100], which reflected the rats' anhedonia levels.

Saccharin Self-Administration
Reward capacity was assessed using the self-administration test, performed as described previously [24]. Briefly, rats were placed on a water restriction schedule for 2-4 days to facilitate training of lever pressing. The rats were trained to self-administer saccharin 0.2% (w/v) in 30-min daily sessions for 2 weeks on a fixed ratio 1 schedule of reinforcement in which each response resulted in the delivery of 0.1 mL of fluid. One lever was paired with the delivery of saccharin as a reward (active lever), whereas the other lever was paired with no reward (inactive lever). At this point, saccharin self-administration training continued until the animals reached stable baseline responding. During the 30 min test sessions, the responses on the active lever and number of saccharin reinforcements were recorded (see the Supplementary Materials for details).

Ethanol Self-Administration Test
Ethanol consumption was assessed using the self-administration test, performed as described previously [24,32] with minor modifications (see the supplementary information for details). Briefly, rats were exposed to intermittent consumption of 0.2% saccharin and 10% ethanol for 3 weeks. After that, rats were placed on a water restriction schedule for 2-4 days to facilitate training of lever pressing. The rats were trained to self-administer saccharin 0.2% (w/v) and 10% ethanol (v/v) in 30 min daily sessions for 2 weeks on a fixed ratio 1 schedule of reinforcement in which each response resulted in the delivery of 0.1 mL of fluid. One lever was paired with the delivery of ethanol as a reward (active lever), whereas the other lever was paired with no reward (inactive lever). At this point, ethanol self-administration training continued until the animals reached a stable level of 10% ethanol responding. During the 30 min test sessions, the responses on the active lever and number of alcohol reinforcements were recorded (see the Supplementary Materials for details).

Forced Swimming Test (FST)
Depressive behaviour was assessed using the FST, performed as described previously [23,26,28]. Briefly, two swimming sessions were conducted: a 15 min pre-test followed 24 h later by a 5 min test. Animals were individually placed in a vertical glass cylinder of 20 cm diameter containing water (25 • C) to a height of 30 cm. The total durations of immobility and swimming behaviour were recorded during the second 5 min test.

Tail Suspension Test (TST)
Depressive behaviour was assessed using the TST, performed as described previously [23,29]. Briefly, rats were hung upside down using an adhesive tape to fix the tail to a rope through an eyebolt at 60 cm. The animal was considered immobile when it was not making any movements of struggling, attempting to catch the adhesive tape, body torsions, or jerks time. The total durations of immobility and mobility behaviour were recorded during the second 5 min test.

Elevated Plus Maze (EPM)
Anxiety behaviour was assessed using the EPM as previously described [33,34]. Briefly, rats were placed on the central platform facing an open arm and allowed to explore the maze for 5 min. The time in open arms and entries to the center were analyzed using the video-tracking software EthovisionXT (see the Supplementary Materials for details).

Open Field Test (OFT)
Anxiety behaviour was assessed using the OFT as previously described [23]. Briefly, the rats were placed in the open field (100 × 100 × 50 cm) and allowed to explore freely for 5 min. Total time spent and interior square inputs were analyzed using EthovisionXT video tracking software.
In the immunohistochemical assays, the cell count was normalized to the total area.

Statistical Analysis
Data are presented as the mean ± standard error of the mean, and sample numbers (n) are indicated in figure legends. All data were analyzed using GraphPad PRISM 8.0 (GraphPad Software, San Diego, CA, USA). For comparing more than two groups, one-way analysis of variance (ANOVA) was performed. Fisher's least significant difference (LSD) comparison post-test was performed only when the F ratio in the one-way ANOVA was statistically significant. Differences were considered statistically significant at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001).
A PCA with varimax rotation was also performed to extract the independent dimensions (i.e., factors) from the C-Fos IR data. Eigenvalue > 1 was chosen as criterion for component extraction and a factor score (i.e., a standardized value indicating the relative position of each animal in each factor) was computed by the regression method (SPPS Statistics 20, IBM Corporation, Armonk, NY, USA). Only measures with a saturation greater than 0.5 in absolute value were included in a factor. The ability of each factor to predict ethanol-seeking behaviour was tested by Pearson's correlations between the factorial score and the rat-score behaviour (see the Supplementary Materials for details).

Behavioural Effects of the Combination of GAL(1-15) and ESC in SPT and Saccharine
Self-Administration Test in Rats 3.1.1. Dose-Response Curve of ESC in SPT and Saccharine Self-Administration Test In SPT, ESC (7.5 mg/kg) induced a significant reduction in sucrose intake (one-way ANOVA, F 2,25 = 4.46, p = 0.02, Fisher's LSD post hoc: p < 0.01) and in sucrose preference (one-way ANOVA, F 2,25 = 4.65, p = 0.01, Fisher s LSD post hoc: p < 0.05, p < 0.01) (Table S1) compared with control animals. However, ESC at 10 mg/kg lacked an effect on sucrose intake or sucrose preference compared with controls animals (Table S1).
In the saccharine self-administration test ( Figure S1), ESC at the doses of 5 mg/kg (p < 0.05) and 7.5 mg/kg (p < 0.001) significantly decreased the number of saccharine reinforcements (one-way ANOVA, F 3,46 = 5.05, p = 0.004, Fisher s LSD post hoc: p < 0.05, p < 0.001; Figure S1B) by 60 and 75%, respectively, and the number of active lever presses (one-way ANOVA, F 3,46 = 4.89, p = 0.004, Fisher s LSD post hoc: p < 0.05, p < 0.001; Figure S1B) compared with the control group. No effects were observed on the number of inactive lever presses at these doses. ESC at a dose of 2.5 mg/kg lacked any effect in the parameters analyzed in the saccharine self-administration test ( Figure S1B).

GAL(1-15) Enhances the Reduction of Reward Capacity of ESC in the SPT and in the Saccharine Self-Administration Test in Rats
The coadministration of ESC (10 mg/kg) and the icv injection of GAL(1-15) (1 nmol) induced a decrease in sucrose intake (one-way ANOVA, F 2,23 = 6.89, p = 0.004, Fisher s LSD post hoc: p < 0.05, p < 0.01; Figure 2A) and in sucrose preference (one-way ANOVA, F 2,23 = 4.80, p = 0.01, Fisher s LSD post hoc: p < 0.05, p < 0.01; Figure 2B) compared with the ESC (10 mg/kg) group in the sucrose preference test, suggesting that GAL(1-15) enhanced the reduction of reward capacity mediated by ESC in this test.

Treatment
Inactive lever presses  Vertical bars represent the mean ± standard error of the mean of sucrose intake (g/kg) and preference (percentage). * p < 0.05; ** p < 0.01, according to one way ANOVA followed by Fisher multiple comparison test. The saccharin self-administration test (C,D): Vertical bars represent mean ± standard error of the mean number of saccharin reinforcements and active lever presses according to the percent baseline during the test period. * p < 0.05 vs. saline + aCSF, according to one-way ANOVA followed by Fisher multiple comparison test. (E): Data represent mean ± standard error of the mean of inactive lever presses according to percent baseline during the test period. There were no differences according to a one-way analysis of variance (ANOVA) between the experimental groups.
In the saccharine self-administration test, the coadministration of threshold doses of GAL(1-15) (1 nmol) and ESC (2.5 mg/kg) induced a strong reduction in the reward capacity of saccharine. Thus, a significant decrease appeared in the number of reinforcements of saccharine self-administration (one-way ANOVA, F 2,21 = 3.85, p = 0.03 Fisher s LSD post hoc: p < 0.05; Figure 2C) and in number of active lever presses (one-way ANOVA, F 2,21 = 3.86, p = 0.04, Fisher s LSD post hoc: p < 0.05; Figure 2D). Although the coadministration of GAL(1-15) + ESC lacked effects on the number of inactive lever presses ( Figure 2E), the high variance showed by the animals could contribute to it.
GAL(1-15) 1 nmol alone had no effect on either the number of saccharine reinforcements or the number of active lever presses in saccharine self-administration [24].

Behavioural Effects of the Combination of GAL(1-15) and ESC in Rats with Chronic Alcohol
Consumption by Self-Administration 3.2.1. The Combination of GAL(1-15) and ESC Induced a Substantial Reduction of Alcohol Intake in the Ethanol Self-Administration Test In the alcohol self-administration test, animals displayed a consistent preference for 10% v/v ethanol (active lever) over no reward (inactive lever) during the FR1 operant responding phase ( Figure 3A).

In Depression Tests Related to Despair, GAL(1-15) Reversed the Adverse ESC-Mediated Effects in Rats with Alcohol Consumption by Self-Administration
In the FST, the subchronic administration of ESC (7.5 mg/kg) in animals with ethanol intake by self-administration induced a significant increase in the immobility time (one-way ANOVA, F 2,19 = 5.86, p = 0.01, Fisher s LSD post hoc: p < 0.01 Figure 4A) and a decrease in the swimming time (one-way ANOVA, F 2,19 = 5.21, p = 0.01, Fisher's LSD post hoc: p < 0.01; Figure 4B) compared with the control group, suggesting an adverse effect of ESC in this test. and Escitalopram (ESC) in the ethanol self-administration Test. ESC was administrated intraperitoneally (ip) 23, 5 and 1 h before the test, and GAL(1-15) 0.3 nmol or aCSF was administered i.c.v 15 min before the test. Saline + aCSF, injected rats were used as control group (n = 7-8 animals/group). (A) Animals displayed a consistent preference for 10% v/v ethanol (active lever) over no reward (inactive lever) during the FR1 operant responding phase. (B,C) Vertical bars represent mean ± standard error of the mean of the number of ethanol reinforcements and active lever presses during the test period. In the figure, the data are represented according to percent baseline during the test period. * p < 0.05 vs. saline + aCSF, ** p < 0.01 vs. saline + aCSF, according to one-way analysis of variance (ANOVA) followed by Fisher's least significance difference test. (D) Data represent mean ± standard error of the mean of inactive lever presses during the test period. There were no differences according to a one-way analysis of variance (ANOVA) between the experimental groups.
However, in the TST, both the administration of ESC (7.5 mg/kg) alone and the coadministration of GAL(1-15) (1 nmol) and ESC (7.5 mg/kg) did not show significant differences in immobility or mobility time.   (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)] and Escitalopram (ESC) in the forced swimming test (FST) and the tail suspension test (TST). ESC was administrated intraperitoneal (ip) 23, 5 and 1 h before the test, and GAL(1-15) 1 nmol or artificial cerebrospinal fluid (aCSF) was administered i.c.v 15 min before the test. Saline + aCSF injected rats were used as the control group (n = 7-8 animals/group). FST (A,B): Vertical bars represent the mean ± standard error of the mean of immobility time and swimming time in FST during the test period. * p < 0.05, ** p < 0.01, according to a one-way analysis of variance (ANOVA) followed by Fisher's least significance difference test. TST (C): Data represent mean ± standard error of the mean of immobility time and mobility time during the test period. There were no differences according to a one-way analysis of variance (ANOVA) between the experimental groups.

Immobility (s) Mobility (s)
In the anxiety tests, no significant effects were found after the coadministration of GAL(1-15) (0.3 nmol) and ESC (2.5 mg/kg) (Table 1) in either the EPM or in the OFT in chronic ethanol exposure rats.

C-Fos Immunohistochemistry Study of GAL(1-15) and ESC Interaction in Rats with Chronic Alcohol Consumption
We analyzed C-Fos IR 90 min after the coadministration of GAL(1-15) (0.3 nmol) and ESC (2.5 mg/kg) in several nuclei involved in depression and AUD-RMTg, LHb, mHb, NAc, PFC-and by double immunohistochemical staining of 5HT/C-Fos in DR or TH/C-Fos in VTA ( Figure 5A,B).

Saline
As seen in Figure 5, in the VTA, the number of C-Fos IR TH cell bodies after icv GAL(1-15) + ESC (one-way ANOVA, F 2,11 = 9.03, p = 0.004, Fisher s LSD post hoc: p < 0.05) and ESC (Fisher s LSD post hoc: p < 0.01) was significantly decreased in comparation with C-Fos IR TH cell bodies in the control group ( Figure 5A).
No significative effects were observed in the rest of the nuclei ( Figure 5). The PCA revealed three independent factors representing the functional brain modules or networks underlying C-Fos IR that explained~80% of the total variance ( Figure 6A,B). The first factor encompassed DR, VTA and RMTg (33.60% of variance explained), the second factor was composed of LHb and mHb (26.10% of variance explained), while the third factor was composed of NAc and CPF (19.02% of variance explained). PCA statistical assumptions were satisfied, allowing its use and interpretation (KMO: 0.349; Bartlett's sphericity test: X 2 (21) = 35.76, p = 0.023).

B.
C.   To determine the relevance of each brain network (factor) in the alcohol self-administration test, we tested for correlations of the factorial scores (FS) for each factor with the number of active lever presses in ethanol self-administration. The number of active lever presses could only be predicted by the scores in Factor 1, composed by DR (FS = −0.811)/VTA (FS = 0.674)/RMTg (FS = 0.810) (r = 0.549; p < 0.05; Figure 6C), indicating that rats that exhibited an increase in C-Fos IR in the VTA/RMTg circuitry were more prone to press the active lever in the alcohol self-administration test.
Additionally, immunohistochemistry of pCREB was performed in NAc. The administration of ESC (2.5 mg/kg) and the coadministration of ESC (2.5 mg/kg) and GAL(1-15) (0.3 nmol) did not show significant differences in the number of pCREB IR cells (Table S2).

Discussion
In the present study, we demonstrated that the combination of GAL(1-15) with ESC induced a substantial reduction of alcohol intake in the ethanol self-administration paradigm. Moreover, GAL(1-15) enhanced the reduction of reward capacity of ESC on different reinforcers such as sucrose or saccharine. The coadministration of GAL(1-15) + ESC significantly decreased the number of C-Fos-IR TH cell bodies in the VTA, and PCA analysis suggested that one functional network, including VTA, RMTg and DR, was involved in these effects on alcohol self-administration.
Interestingly in rats with alcohol consumption by self-administration, GAL(1-15) reversed the adverse ESC-mediated effects in depression-related behavioural test FST, confirming that the combination GAL(1-15) + ESC also improved the depressive symptoms induced by the ESC.
Not only in the operant models but also in the non-operant model using the natural reinforcer sucrose (SPT) [38,39], the combination of GAL(1-15) and ESC induced a significant reduction in sucrose preference and sucrose intake, confirming a substantial effect for this cotreatment in the reward system.
The fact that the GAL(1-15) and ESC combination can modulate the reward system with both natural and artificial reinforcers such as alcohol, in addition to improving depressive symptoms in an animal model of depression such as OBX rats [29] opens up the possibility to use this combination as augmentation therapy in the depression and AUD comorbidity.
The role of neurotransmitter 5-HT as a modulator of reward function is widely described in the literature. Serotonin reduces the reinforcing properties of food, saccharin, alcohol, psychostimulants, and direct electrical brain stimulation [13,[40][41][42][43]. Treatment with SSRIs or the 5-HT releaser dexfenfluramine consistently decreases instrumental responding for primary reinforcers such as food, drugs of abuse [44], and brain stimulation reward [45,46]. Moreover, both in SERT-KO mice and after acute administration of citalopram, the primary reinforcer saccharine is reduced [47]. Recent optogenetic studies also found that combining stimulation of DRN 5-HT neurons with a low dose of the SSRI citalopram reduced the operant responding by saccharine [40].
The mechanism involved in the 5-HT effect in the reward system affects the DA system. The serotoninergic neurons from DR innervate the VTA and inhibit mesolimbic DA activity [40]. Additionally, in the NAc, the MSNs neurons synapse onto DR 5-HT neurons, directly influencing reward processes [48,49]. Several studies showed that SSRIs, including ESC, reduced DA neuronal activity in the VTA and DA release in the striatum [50][51][52][53].
The reduction of DA neuronal activity is probably related to a serotonergic-dopaminergic interaction in the mesolimbic system via 5-HT2C receptors [53,54].
Our results are in agreement with these works since ESC reduced the reward capacity in the operant and non-operant models, confirming that 5-HT decreases the reward function. Moreover, the coadministration of GAL(1-15) + ESC produced a significant decrease in the number of C-Fos-IR TH cell bodies in the VTA, and the analysis of the relevance of the different brain networks in the alcohol self-administration test indicated that rats that exhibited an increase in C-Fos IR in the VTA/RMTg circuitry were more prone to press the active lever in the alcohol self-administration test, confirming the importance of VTA in the GAL(1-15) + ESC-mediated effect.
It is of high interest that the effect of GAL(1-15) and ESC coadministration was found in the FST, a depression behavioural test related with despair, in the animals with alcohol intake by self-administration. Our results indicated that ESC administration alone induced an increase in immobility and a decrease in swimming, suggesting a worsening of depressive symptoms. However, the coadministration of ESC with GAL(1-15) reversed the adverse effects induced by ESC alone in the FST.
ESC effects in naive rats show a variability of response: no effect [55], or a decrease [56] of immobility in the FST. Interestingly, chronic ethanol intake alters 5HT1A receptor density and/or expression in the brain [57][58][59], showing an increase in the density and expression of somatodendritic 5HT1A autoreceptor [57] and a 5HT1A autoreceptor supersensitivity [60] in the DR. This increase in the density and functional sensitivity of the 5HT1A autoreceptor could explain the pro-depressive effects of ESC in the FST; ESC through the SERT lock raises the central tone of 5-HT, which increases the activation-hypersensitive autoreceptors induced by the alcohol and produces a decrease in the 5-HT released.
We have described the GAL(1-15) ability to modulate the 5HT1A receptor characteristics and expression level [26]. Thus, in the DR, GAL(1-15) reduced the density of 5HT1A autoreceptor and its mRNA levels, suggesting an enhancement in the firing rate of the ascending 5HT DR neurons [26]. This mechanism could underlie the enhancement of ESC by GAL (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) in the FST in the present work. GAL(1-15) could modulate the alcohol-induced increase in density and functional sensitivity at somatodendritic 5HT1A receptors in DR, thus avoiding the depressive adverse effects shown by ESC in alcohol-consuming rats. However, a detailed study of this mechanism should be carried out in the future.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10020412/s1. Figure S1. Effect of the administration of GAL(1-15) and ESC in the saccharin self-administration test; Table S1. ESC dose response curve in the sucrose preference test (SPT); Table S2. Effect of the administration of GAL(1-15) and ESC on pCREB expression.

Data Availability Statement:
The data presented in this study will be openly available in RIUMA-University of Malaga once the manuscript is accepted for publication.

Conflicts of Interest:
The authors declare no conflict of interest.