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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Addict Biol. 2018 Oct 17;24(5):981–993. doi: 10.1111/adb.12671

phMRI, neurochemical and behavioral responses to psychostimulants distinguishing genetically selected alcohol-preferring from genetically heterogenous rats.

A Bifone 1, A Gozzi 1, A Cippitelli 2, A Matzeu 3, E Domi 2, H Li 2, G Scuppa 1,2, N Cannella 2, M Ubaldi 2, F Weiss 3, Ciccocioppo 1,*
PMCID: PMC6697752  NIHMSID: NIHMS983538  PMID: 30328656

Abstract

Alcoholism is often associated with other forms of drug abuse, suggesting that innate predisposing factors may confer vulnerability to addiction to diverse substances. However, the neurobiological bases of these factors remain unknown. Here, we have used a combination of imaging, neurochemistry and behavioral techniques to investigate responses to the psychostimulant amphetamine in Marchigian-Sardinian (msP) alcohol-preferring rats, a model of vulnerability to alcoholism. Specifically, we employed pharmacological Magnetic Resonance Imaging (phMRI) to investigate the neural circuits engaged by amphetamine challenge, and to relate functional reactivity to neurochemical and behavioral responses. Moreover, we studied self-administration of cocaine in the msP rats. We found stronger functional responses in the extended amygdala, alongside with increased release of dopamine in the nucleus accumbens shell and augmented vertical locomotor activity compared to controls. Wistar and msP rats did not differ in operant cocaine self-administration under short access (2hrs) conditions, but msP rats exhibited a higher propensity to escalate drug intake following long access (6 hrs). Our findings suggest that neurobiological and genetic mechanisms that convey vulnerability to excessive alcohol drinking also facilitate the transition from psychostimulants use to abuse.

Keywords: Reward, addiction, phMRI

Introduction

The Marchigian Sardinian alcohol-preferring (msP) rat is an established selection-based model for the investigation of the neurobiology of alcoholism (Ciccocioppo et al. 2006). This line of rats closely mimics several fundamental aspects of the human disease such as the occurrence of binge-like ethanol drinking (Ciccocioppo et al. 2006), psychological withdrawal symptoms (Ciccocioppo et al. 1999), escalating alcohol intake upon abstinence, and high-vulnerability to stress-mediated relapse (Hansson et al. 2006). Of significance, this model also reproduces important co-morbid states pervasively associated with alcohol use disorder, such as increased sensitivity to stress, anxious phenotype and depressive-like symptoms (Ciccocioppo et al 1999; Ciccocioppo et al. 2006; Cippitelli et al 2015; Hansson et al. 2006). Based on these findings, we proposed that the msP rat is an animal model endowed with substantial predictive, face and construct validity, and therefore provides valuable tool to generate addiction-relevant data with significant translational value (Ciccocioppo et al. 2012). A highly important, but underexplored phenomenon in laboratory animals, including msP rats, is that alcohol consumption in humans often co-occurs with other drugs of abuse. For example, concomitant use of alcoholic beverages and nicotine is nearly universal (DiFranza, Guerrera 1990; Miller, Gold 1998). Among illicit drugs, co-abuse of cocaine and other psychostimulants with alcohol is a very common occurrence (Grant, Harford 1990).

In humans, it is estimated that predisposing genetic factors account for about 60 % to the actual development of alcohol use disorder (Schuckit 1998; Prescott, Kendler 1999). Several genetic traits associated with alcohol abuse are associated also with other forms of dependence indicating that common innate factors may be responsible for predisposition to addiction in general. If true, one would predict that animals genetically selected for excessive alcohol drinking are more vulnerable to the addictive actions of other drugs of abuse as well. This hypothesis has found support in earlier studies with P rats, another rodent line genetically selected for excessive alcohol consumption. In fact, it has been shown that P rats are more sensitive to the rewarding effects of nicotine and self-administer larger amounts of the substance compared to Wistar controls (Hauser et al. 2014). To our knowledge, no studies have investigated the propensity of alcohol preferring rats to also show heightened preference or intake of psychostimulants such as amphetamine or cocaine.

Animal models, including genetically selected rat lines, offer the opportunity to conduct studies under strictly controlled conditions, and provide an important tool for identifying genetic and environmental factors that influence alcohol- and possibly polydrug- abuse related traits. However, one of the challenges for the definition of translational phenotypes is to employ appropriate techniques permitting valid correlation of preclinical neurobiological findings with specific aspects of the human syndrome.

Neuroimaging methods have been extensively applied to study the human brain and its structural and functional organization in healthy and pathological states. Imaging techniques permit exploration of endophenotypes that are more proximal to the biological mechanisms underlying the risk for developing drug abuse disorders. An important advantage of the neuroimaging approach is that the output does not rely on subjective reports of an effect, but represents a measure of a biologically-based expression of the phenotype. Recent developments have extended neuroimaging approaches to animal models (Bifone, Gozzi 2011, 2012), thus paving the way toward the translational use of neuroimaging techniques to bridge clinical and preclinical research.

In the present study, we conducted a series of experiments aimed at two major objectives. Firstly, using Magnetic Resonance Imaging (MRI), a translational technique that enables examination of drug-induced brain activation patterns, we evaluated the response to an amphetamine challenge in msP rats compared to controls. As a main fMRI parameter we analysed the relative cerebral blood volume (rCBV) which reflect neuronal activation in brain areas. Secondly, we intended to establish whether genetic selection for excessive alcohol drinking has co-segregated traits responsible for the predisposition to develop abuse of other addictive drugs. We decided to investigate psychostimulants because they are, together with nicotine, the drugs most frequently used in association with alcohol (Grant, Harford 1990)(Gossop et al., 2006; Higgins et al., 1994). For this purpose we analyzed neurochemical and behavioral responses of msP and Wistar rats produced by amphetamine and cocaine. For behavioural analysis, in addition to open field locomotor activity, we used operant self-administration Fixed Ratio (FR) and Progressive Ratio (PR) paradigms to evaluate the reinforcing effect of psychostimulants.

Materials and Methods

Animals

Heterogeneous male Wistar (Charles River, Feld, Germany,) and male genetically selected alcohol-preferring Marchigian Sardinian (msP) rats were used (Ciccocioppo et al. 2006). MsP rats were bred at the School of Pharmacy of the University of Camerino (Marche, Italy). At the time of the experiments the rats’ body weight ranged between 350 and 400 g. They were pair-housed in plexiglass cages on a reverse 12:12 h light/dark cycle (lights off at 9:30 a.m.), temperature of 20–22°C and humidity of 45–55%. Rats were offered free access to tap water and food pellets (4RF18, Mucedola, Settimo Milanese, Italy). All procedures were conducted in adherence with the European Community Council Directive for Care and the Use of Laboratory Animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Drugs

D-amphetamine sulphate (Sigma), the active amphetamine enantiomer, was dissolved in sterile saline and was administered intravenously (IV) in the phMRI experiments. For microdialysis and open field experiments, racemic amphetamine sulphate (Sigma) was given intraperitoneally (IP). In experiments where racemic amphetamine was used the dose was double because half of it is the inactive enantiomer (L-amphetamine). Cocaine hydrochloride (Johnson Matthey, Edinburgh, UK) was dissolved in sterile physiological solution at a concentration of 0.075, 0.250 and 0.750 mg/0.1 ml and given IV for the self-administration experiments.

Magnetic Resonance Imaging.

Animal preparation and MRI acquisition parameters have been described in great detail (Ferrari et al. 2013). Briefly, msP rats (N=22) and outbred Wistar rats (N=26), were anaesthetized with 3% halothane, tracheotomized and artificially ventilated with a mechanical respirator. After surgery, halothane level was set to 0.8%, an anesthetic regimen shown to preserve phMRI responses to d-amphetamine (Gozzi et al. 2008). Arterial blood gases (paCO2 and paO2) were measured before and after bCBV measurement and ventilation parameters were adjusted to keep gas levels within physiological range (Ferrari et al. 2012). Anatomical and bCBV time series were acquired on a Bruker Biospec 4.7 Tesla scanner. The body temperature of all subjects was maintained within physiological range (37±0.8 °C) throughout the experiment by using a water heating system incorporated in the stereotactic holder.

T2-weighted anatomical volumes were acquired using a RARE sequence (TEeff = 72 ms, RARE factor 8, FOV 40mm, 256×256matrix, 20 × 1mm slices) followed by time series acquisition (TR = 2700 ms, TEeff = 111 ms, dt=60 s) with the same spatial coverage but lower in-plane resolution (128 × 128). Following five reference images, 1.5 ml/kg of the contrast agent Molday Ion (BioPal, Worcester, USA) were injected to make the MRI signal changes sensitive to relative cerebral blood volume (Schwarz et al. 2003; Schwarz et al. 2007; Gozzi et al. 2013). After an equilibration period of 15 min each subject received an acute intravenous challenge with d-amphetamine (0.5 mg/kg) or saline. Starting one minute after injection images were acquired again.

Microdialysis

Surgery.

One week before the microdialysis test animals were surgically implanted with a guide cannula (Plastics One, Roanoke, VA; outer diameter, 300μm) aimed at the nucleus accumbes (NAC) shell. The guide cannula was implanted unilaterally, in the right or the left hemisphere and secured with stainless steel skull screws and dental cement. The NAC shell coordinates were: AP +1.6 mm, ML ±0.8 mm from bregma; DV −5.8 mm from dura, according to Paxinos and Watson (Paxinos, Watson 1986). The microdialysis site was located between ventral coordinates V −5.8 and V −7.8 from dura (2 mm below the tip of the guide cannulae).

Microdialysis.

Microdialysis probes were made in-house and constructed by inserting two pieces of fused silica tubing (OD 106.0 μm, ID 40.0 μm; Polymicro Technologies) into a 5-mm length of regenerated cellulose dialysis membrane (OD 280 μm, ID 200 μm, cutoff 13kD; Spectrum Laboratories, Inc.). The active length of dialysis membrane was restricted to 2 mm. The probes were inserted under brief isoflurane anesthesia (< 5 min) 12–16 h before the start of testing. The probes were perfused with artificial cerebrospinal fluid (145 mM NaCl, 2.8 mM KCl, 1.2 mM MgCl2, 1.2 mM CaCl2, 0.25 mM Ascorbic Acid, 5.4 mM D-glucose, pH 7.2–7.4) at a rate of 0.6 μl/min using a syringe pump (Harvard 22 Syringe Pump, Holliston, MA). Dialysate was collected into 250 μl (Fisherbrand) microfraction vials every 10 min. Samples were immediately frozen on dry ice and stored at - 80°C until assayed. After 30 min collection of basal samples, rats were monitored for 60 min following an i.p. vehicle injection and subsequently for 90 min following an amphetamine sulphate injection (1 mg/kg, IP). One group of Wistar (n=7) and one group of msP (n=7) were used. Samples from 12 animals (6 per line) were used for dopamine detection, and 14 (7 per line) for glutamate and GABA.

Dopamine HPLC detection.

Dialysate dopamine concentration was measured by reverse-phase HPLC coupled with electrochemical detection. The mobile phase consisted of 100 mM NaH2PO4, 59.41 mM Na2HPO4, 0.75% methanol, 0.134 mM EDTA, 3.27 mM Sodium 1-decanesulfonate (pH 6.0). The analytical system consisted of an isocratic pump, an electrochemical detector (HPLC-ECD Stand Alone System, HTEC-500, EICOM corporation, San Diego, CA) with a graphite working electrode operating at + 400 mV vs. Ag/AgCl (Eicom corporation, San Diego, CA) and a reverse phase C18 column (PP-ODS 4.6 × 30 mm; EICOM corporation, San Diego, CA). The mobile phase was pumped through the column at a flow rate of 400 μl/min. Dialysate samples were injected onto the column at a volume of 2 μl/min via a valve with a 5 μl loop (Rheodyne, L.P., Rohnert Park, CA). The detection limit for dopamine was 1.5 nM with a signal noise/ratio of 3/1.

Glutamate and GABA HPLC detection.

Dialysis samples were analyzed with HPLC to determine glutamate and GABA contents. Glutamate and GABA were measured simultaneously by reverse-phase HPLC (Agilent 1100 Series with a 100×3 mm, C18, 5 μm particle size column, Thermo SCIENTIFIC) coupled to a fluorescent detector (xenon flash lamp, Agilent 11000 Series Fluorescence Detectors, excitation wavelength 419 nm and emission wavelength 493 nm) using Agilent ChemStation Plus software. Pre-column derivatization required for detection of the amino acids was conducted using 15 μl of borate buffer (911 μl of borate solution 40 mM plus 89 μl of KCN solution 43 mM), 2 μl sample/standard and 3 μl NDA solution 5 mM, with a final volume of 20 μl. Gradient elution was used to separate the mixture of amino acids. Mobile phase A consisted of 100 mM sodium acetate water solution at pH 6.00, while mobile phase B was a 100 mM sodium acetate methanol solution. Samples were injected onto the column at a volume of 18 μl via an automated injector (Agilent 1100 Series Autosampler). The detection limit for glutamate and GABA was 3 nM with a signal noise/ratio of 3/1.

Histology:

Upon completion of the microdialysis experiment, rats were euthanized with 100% carbon dioxide. Brains were removed, frozen with 2-Methylbutane (Sigma-Aldrich) and dry ice, and subsequently cut with a Microtome (Leica, SM 200R) into 55 μm coronal sections. Probe placements within the NAC shell were verified by visual inspection of probe tracts (Fig. 1).

Figure 1.

Figure 1.

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Coronal brain section detailing locations of the microdialysis probes within the Nac shell.

Open field test

Automated open field (OF) locomotor activity chambers (Med Associates, VT) were used. The system consisted of square (43.2 cm × 43.2 cm × 30.5 cm) plexiglass cages equipped with 10 equally spaced infrared light beams and placed in a dimly illuminated room. Beams breaks were recorded to monitor total distance traveled (horizontal activity), and rearings (vertical activity). Wistar (n=24) and msP (n=24) rats were randomly divided into three groups each (n=8/group). The experiment was conducted as follows: for two consecutive days, rats were familiarized with the ip injection procedure and acclimated to the open field arena for 20 minutes. The following day rats were subjected to a third 20 min open field session, immediately followed by injection of amphetamine (0, 1 and 2 mg/kg, IP) and return to the home cages. After 20 min rats were re-introduced to the open field and locomotor activity was monitored for an additional 20-min.

Operant self-administration

Intravenous (IV) Catheterization:

Rats were anesthetized by intramuscular injection of 100–150 μl of a solution containing tiletamine chlorohydrate, (58.17 mg/ml) and zolazepam hydrochloride (57.5 mg/ml). For IV surgery, incisions were made to expose the right jugular vein. A catheter made from micro-renathane tubing (I.D = 0.020 inches, O.D = 0.037 inches) was subcutaneously passed from the animal’s back to the jugular vein. After insertion into the vein, the proximal end of the catheter was anchored with surgical silk to the muscles underlying the vein. The distal end of the catheter was attached to a stainless steel cannula bent at a 90° angle. The cannula was inserted into a support made by dental cement on the back of the animals and was covered with a plastic cap. For one week after surgery, the rats were treated daily with 0.2 ml of the antibiotic sodium cefotaxime (262 mg/ml). For the duration of the experiments, the catheters were flushed daily with 0.2–0.3 ml of heparinized saline solution. Body weight was monitored every day, and catheter patency was confirmed approximately every 7 days with an injection of 0.2–0.3 ml of thiopental sodium (250 mg/ml) solution. Patency of the catheter was assumed if there was an immediate loss of reflexes. Self-administration experiments began 1 week after the post-surgery recovery.

Self-administration apparatus:

The self-administration stations consisted of operant conditioning chambers (Med Associate Inc) enclosed in sound attenuating, ventilated environmental cubicles. Each chamber was equipped with two retractable levers located in the front panel of the chamber. A plastic tube that was connected to the catheter before the beginning of the session delivered cocaine. An infusion pump was activated by responses on the right (active) lever, while responses on the left (inactive) lever were recorded but did not result in any programmed consequences. Activation of the pump resulted in a delivery of 0.1 ml of fluid. An IBM compatible computer controlled the delivery of cocaine solution and recording of the behavioural data.

Fixed Ratio 5 (FR) and progressive ratio (PR) cocaine self-administration:

Following one week of recovery from surgery msP (n=7) and Wistar rats (n=8) were trained to self-administer cocaine under a FR-5 schedule of reinforcement; every 5 active lever presses resulted in the delivery of one cocaine training dose (0.25 mg/0.1 ml, IV) Once a stable baseline of responding was reached, msP and Wistar rats were switched to different concentrations of cocaine (0.075 and 0.750 mg/0.1 ml infusion) in a counterbalanced order so that all animals received the two different concentrations for five consecutive days each. At the end of 0.25 mg/inf training animals were subjected to the first PR experiment. PR test was replicated after rats were were switchedand maintained for 5 days to the lowest (0.075 mg/0.1ml infusion) and the highest (0.750 mg/0.1ml infusion) cocaine doses. The break point (BP), defined as the last ratio completed by the animal to obtain one cocaine infusion, was used as a measure of cocaine reinforcement (Roberts, Richardson 1992). For this purpose, the response requirement (i.e., the number of lever responses or the ratio required to receive one dose of cocaine) was increased as follows: 5, 11, 18, 26, 35, 45, 56, 68, 82, 98, 116, 136, 158, 182, 208, 236, 268, 304. Each cocaine-reinforced response resulted in the house light being turned on for 20 s, whereas sessions were terminated when more than 2 h had elapsed since the last reinforced response. The experiment was conducted in parallel for the msP and Wistar rat lines. After having switched the animals from the dose 0.25 to the others, two subjects lost the catheter, therefore 0.075 and 0.75 doses were tested in 6 msP and 6 Wistar rats.

Cocaine self-administration under long vs short access condition:

Wistar and msP rats were first trained to self-administer cocaine in1 h daily sessions. Initially, training was conducted on a FR1 schedule at the 0.25 mg cocaine dose. Subsequently rats were switched to an FR5 contingency. Once stable FR5 baseline responding was reached, both Wistar and msP rats were divided into 2 groups. One group of msP (N= 9) and one group of Wistar (N=9) rats was maintained 1 h of daily self-administration (short access; Sha), whereas the second group of msP (N= 9) and Wistar (N=9) was switched to 6 h of daily self-administration session (long access; Lga). Self-administration under Sha and Lga conditions continued until rats showed stable baselines of responding. To measure escalation of cocaine self-administration in the Lga compared to Sha condition, the number of reinforced responses during the first hour in both groups was compared throughout the self-administration period.

In self-administration experiments cocaine was used as a reinforcer because its short half-life allows the possibility to analyse the dose-effect relationship for its reinforcing effect and also the escalation under LgA condition. These parameters are critical to evaluate whether differences in lever pressing between msP and Wistar rats may be due to a higher or lower sensitivity to the reinforcing action of the psychostimulant. This kind of analysis is extremely difficult with amphetamine because, due to its longer half-life, at intermediate/high doses it accumulates causing marked pharmacokinetic-related drop in lever response, which limits the interpretation of the data.

Statistical Analysis

Pharmacological MRI data analysis:

rCBV time series image data for each experiment were analyzed within the framework of the general linear model (GLM) to obtain Z statistic maps (Worsley et al. 1992) guiding the selection of activated regions for subsequent volume of interest (VOI)–based quantification and comparison of efficacy of treatments. The procedure used to calculate amphetamine-induced phMRI maps has been previously described (Gozzi, et al. 2011a; Gozzi et al. 2011b). Briefly, rCBV time series were spatially normalized to a stereotaxic rat brain MRI template set (Schwarz et al. 2006) and signal intensity was converted into relative cerebral blood volume (rCBV(t)) on a pixel-wise basis (Gozzi et al. 2011a). Individual subjects in each study were spatially normalized by a 9 degree of freedom affine transformation mapping their T2-weighted anatomical images to a stereotaxic rat brain MRI template set and applying the resulting transformation matrix to the accompanying rCBV time series. Image based time series analysis was carried out using FEAT (FMRI Expert Analysis Tool) Version 5.63, part of FSL (FMRIB’s Software Library, www.fmrib.ox.ac.uk/fsl) with 0.8 mm spatial smoothing (≈ 2.5 × in-plane voxel dimension) and using a boxcar function capturing the temporal profile of the signal change induced by the amphetamine challenge in each group (Schwarz et al. 2006). Higher-level group comparisons were carried out using FLAME (FMRIB’s Local Analysis of Mixed Effects); Z (Gaussianised T/F) statistic images were thresholded using clusters determined by Z>1.9 and a corrected cluster significance threshold of p=0.001.

Behavioral data analysis:

Locomotor activity was analyzed by a two-way ANOVA with rat line and treatment (doses) as between factors. Cocaine self-administration was evaluated by two-way ANOVA with one between factor (rat line) and one within factor (time; days). Different cocaine concentrations were analyzed separately. For the progressive ratio experiment, data were analyzed with the Man-Withney non parametric test because the break point measure violated the assumption of normal distribution. Also, in this case, different cocaine concentrations were analyzed separately. For strain differences in ShA vs. LgA cocaine self-administration, data were analyzed with a two-way ANOVA with one between factor (ShA vs. LgA) and one within factor (time; days).

Microdialysis data analysis:

For the microdialysis experiment, dopamine, glutamate and GABA data were analyzed separately. The averages of baseline concentrations of dopamine, glutamate or GABA in msP and Wistar rats were analyzed by t-tests. Time courses of dopamine, glutamate and GABA fluctuations were analyzed by two-way ANOVA with strain (2 levels) as the between-group factor and time (18 levels) as the within-group factor. Where appropriate, Bonferroni tests were used for post-hoc comparison. In each strain the fluctuations of dopamine, glutamate and GABA concentrations were analyzed by one-way repeated measures ANOVA, followed by Newman-Keuls post-hoc tests.

RESULTS

msP rats display increased phMRI response to amphetamine in the extended amygdala network

For a hypothesis-free analysis of brain regions that exhibit differential pharmacological response to dopamine-mimetic drugs, phMRI was applied to map the acute response produced by behaviorally efficacious dose of d-amphetamine, a drug that stimulates synaptic dopamine release (Knutson, Gibbs 2007). Consistent with previous studies (Schwarz et al. 2004), d-amphetamine, produced robust, widespread and sustained rCBV increases in cortical and subcortical regions. Genetic-related differences in d-amphetamine, responses were observed in several brain areas (Figure 2), including robustly attenuated responses in cortical areas, as well as increased activation in regions of the anterior cingulate in msP compared to Wistar rats.

Figure 2.

Figure 2.

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Anatomical distribution of brain regions exhibiting significantly reduced (blue scale) or increased (red-yellow scale) phMRI responses to d-amphetamine (0.5 mg/kg) in msP compared to Wistar rats (z>1.9, p=0.001). [Abbreviations: VL: ventrolateral thalamus; CeA: central nucleus of the amygdala; Rs: restrosplenial cortex; AD: anterodorsal thalamus; Cg: cingulate cortex; BNST: bed nucleus of the stria terminalis; S1: sensory cortex 1; M1: motor cortex 1; mPFC: medial prefrontal cortex; AcbSh: shell of the nucleus accumbens.]

Compared to Wistars, in msP rats, increased functional reactivity was detected in the prelimbic cortex, dorsal striatum, and the extended amygdala network, with clear involvement of all the major components of this circuit including the central nucleus of the amygdala, bed nucleus of stria terminalis and shell of the nucleus accumbens (Figure 2). Time courses of the rCBV signal for some brain regions showing higher, lower or no difference in rCBV between strains are also reported (Figure 3).

Figure 3.

Figure 3.

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PhMRI time courses following d-amphetamine challenge in Wistar and msP rats in four representative brain structures. t=0 indicates the time of d-amphetamine injection. Data are plotted as mean (±SEM) within each group.

Following an amphetamine challenge msP revealed a higher peak in extracellular DA levels in the NACC shell compared to Wistar rats

Baseline levels.

Analysis of the mean (±SEM) basal concentrations of extracellular dopamine, glutamate and GABA at baseline (not corrected for in vitro dialysis probe recovery) in NAC shell did not show significant differences between the msP and Wistar lines (dopamine: t10 = 0.6908, ns; glutamate: t12 = 0.3007, ns; GABA: t12 = 0.2039, ns; data not shown).

Dopamine.

An amphetamine challenge (1 mg/kg, IP) increased extracellular DA in NAC shell in both Wistar and msP rats. Fluctuations of local concentration of extracellular DA were found to depend on Time (F17,170 = 85.09; p<0.001) and a Time × Strain Interaction (F17,170 = 6.092; p<0.001), but not on Strain (F1,170 = 3.143; ns). Additional analyses revealed that administration of vehicle did not have a significant effect on extracellular DA in the NAC shell in both lines of rats, while DA levels were significantly elevated following injection of amphetamine sulphate for both rat lines as measured 10 min following injection in msP and 20 min after injection in Wistar rats (msP: F17,170 = 9.085, p<0.001; Wistar: F17,170 = 10.72, p<0.001). Bonferroni post-hoc test revealed that the increase in DA 20 and 30 min after injection was significantly higher in msP than in Wistar rats (Fig. 4).

Figure 4.

Figure 4.

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Effect of an amphetamine (1 mg/kg i.p.) challenge on dialysate DA levels in the Nac shell in msP and Wistar rats. Amphetamine enhanced extracellular DA levels in both rat lines, however the increase was significantly higher in msP rats. *p<0.05 msP vs Wistar rats.

Glutamate.

Amphetamine did not modify extracellular glutamate in the NAC shell in either Wistar or msP rats. Local concentration of extracellular glutamate fluctuated only in a time-dependent manner (F17, 204 = 1.873, p<0.05; data not shown).

GABA.

A challenge of amphetamine did not modify extracellular GABA in NAC shell in either line of rats. Local concentrations of extracellular GABA fluctuated only in a manner dependent on the Time (F17,204 = 3.030, p<0.001; data not shown).

MsP rats showed higher vertical locomotor behavior in response to amphetamine challenge

In the open field test, horizontal and vertical activity were monitored. ANOVA revealed a significant effect of amphetamine treatment in horizontal distance travelled (F2,42=26.67, p<0.001). No differences between msP and Wistar rats were detected (F1,42 = 0.0, ns]. However, a significant Line × Amphetamine Treatment interaction was detected (F2,42=3.5 p<0.05). As shown in Figure 5A, Newman-Keuls post hoc tests revealed a significant increase in the distance travelled following both low and high amphetamine doses in both rat lines with a different trend as function of the dose, an increase in msP rats and a decrease in Wistars, respectively. ANOVA also revealed a significant effect of amphetamine treatment on vertical counts (F2,42=29.55, p<0.001), with a significant line difference (F1,42=13.27, p<0.001). No significant Treatment × Line interaction was registered (F2,42=1.57, ns). As shown in Figure 5B, amphetamine produced a more robust vertical activity in msP compared to Wistar rats.

Figure 5.

Figure 5.

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Effect of two different doses (1 or 2 mg/kg) of amphetamine on locomotor activity in Wistar and msP rats assessed in the open field test. A) Both amphetamine doses significantly increased the distance travelled in both rat lines. B) Amphetamine (1 mg/kg) significantly increased vertical activity in msP but not Wistar rats; amphetamine (2 mg/kg) increased vertical activity in both lines. Values represent the mean (± SEM). ** p<0.01, *** p<0.001 vs vehicle and +++p<0.001 difference between rat lines

Compared to Wistars, msP rats have reduced response to a low cocaine concentration under PR schedule of reinforcement.

Under the FR5 contingency, ANOVA revealed no significant differences between msP and Wistar rats in response to different cocaine concentrations (0.075 mg/inf: F1,10=3.5, NS; 0.25 mg/inf: F1,13=3.6; NS; 0.750 mg/inf: F1,10=3.2; NS). As shown in Figure. 6A–C, msP and Wistar rats achieved the same level of cocaine infusions at all doses tested. On the other hand, statistical analysis revealed significant differences between rat lines under the PR contingency. As shown in Figure 6D–F, the Mann-Whitney U test revealed a significantly higher BP in Wistar rats at the 0.075 mg/inf cocaine concentration (U=2.6, p<0.05). No differences were detected at higher concentrations.

Figure 6.

Figure 6.

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Self-administration of cocaine at different concentration in msP and Wistar rats. A, B, C: no differences were observed in cocaine self-administration under a FR5 schedule of reinforcement between msP and Wistar rats. D,E,F: difference in cocaine self-administration under a progressive ratio schedule of reinforcement between msP and Wistar rats were observed only at 0.075 mg/inf. Values represent the mean (±SEM). * p<0.05 msP vs Wistar.

Compared to Wistars, msP rats show higher propensity to escalate cocaine self-administration following long access exposure.

In the ShA condition, overall ANOVA showed no difference in cocaine-reinforced responding between rat lines (F1,16=1.25, NS). Similarly, no line differences were detected in total responses in the LgA condition (F1,16=0.02, NS). When escalation of cocaine self-administration was analyzed by comparing cocaine-reinforced responses during the 1h ShA and the first hour of the LgA session, ANOVA revealed a significant line difference (F 1,16 =40.88, p<0.001, a significant effect of days (F22,16=24.73, p<0.001) and a significant line × days interaction (F22,704=34.80, p<0.001). Comparing the first hour of the LgA with that of the ShA session, we found that msP rats escalated from 13.9±1.4 to 25±0.8, whereas Wistars increased their lever pressing from 17.8±1.2 to 22.6±2.0. In particular, as shown in Figure 7 which depicts the Δ difference (number of responses in the first hour of LgA minus responses of the 1 h ShA session), throughout the self-administration training msP rats showed a higher propensity to escalate cocaine intake compared to Wistar rats.

Figure 7.

Figure 7.

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Escalation of cocaine intake over 21 sessions in msP and Wistar rats. For each session the Δ difference was calculated as the number of lever presses in the first hour of LgA self-administration minus lever presses during the 1 h ShA session in msP (filled circles) and Wistar rats (open circles).

Discussion

The brain imaging results revealed differential pharmacological responses to acute d-amphetamine infusion in msP compared to Wistar rats. Specifically, a number of genetic-related differences in d-amphetamine responses were observed, with msP rats showing a reduced response to the drug in cortical areas, including sensory and motor areas. Conversely, increased functional reactivity was observed in the prelimbic cortex, dorsal striatum and, most notably, in the extended amygdala networks, including the central nucleus of the amygdala, bed nucleus of stria terminalis and NAC shell. To a large extent, these areas represent terminal regions innervated by the mesocorticolimbic dopaminergic system that originates in the VTA (Yetnikoff et al. 2014). Based on this neuroanatomical consideration, we hypothesized that enhanced activation in these areas is related to a heightened response of DA neurons to d-amphetamine in msP compared to Wistar rats. To test this prediction, we monitored extracellular DA levels the NAC shell in response to an amphetamine challenge. The results revealed that, at baseline, the concentration of DA in msP and Wistar rats was similar. However, following amphetamine injection, peak DA concentrations were significantly higher in msP rats. In contrast, extracellular GABA and glutamate levels did not differ between rat lines either under baseline conditions or following stimulation. At the behavioral level, DA transmission in the ventral striatum is known to regulate locomotor activity and reward (Koob 1992; Stewart, Badiani 1993; Pierce, Kalivas 1997). We anticipated therefore that amphetamine would produce greater motor activation in msP rats compared to Wistars. An additional prediction was that msP rats would be more prone to self-administer a psychostimulant. The locomotor activity data partially confirmed our hypothesis because, even though horizontal activity was comparable in the two rat lines, we found that vertical activity, a measure of exploration, was higher in msP than in Wistar rats. We next explored the animals’ behavior in response to IV cocaine by testing operant self-administration under FR-5 and PR schedules of reinforcement at low, intermediate and high drug doses. Contrary to our expectation, there were no differences in cocaine intake at all three doses tested. However, at the lowest cocaine dose, the BP for cocaine was significantly lower in msP than Wistar rats, possibly indicating reduced sensitivity to the reinforcing effects of low psychostimulant doses in this genetically selected rat line. This finding is in apparent contradiction with the microdialysis findings that show increased extracellular DA levels in msP rats following amphetamine injection, which should reflect an enhanced reward response to stimulants. On the other hand, several lines of evidence show that msP rats have genetic and behavioral characteristics resembling a post-dependent phenotype, at least with regard to alcohol (Hansson et al. 2006). These include increased expression and functionality of the extrahypothalamic corticotropin releasing factor receptor 1 (CRFR1) system, enhanced anxiety and depressive-like behaviors mimicking the typical dysphoric state associated with drug withdrawal (Hansson et al. 2006; Hansson et al. 2007). Moreover, in sP rats, a progenitor line from which msP have been derived, a marked reduction in the expression of D1 and D2 receptors in nucleus accumbens was reported (Stefanini et al. 1992; De Montis et al. 1993). This lack of DA receptor function reflects results obtained in human drug addicts who show a hypodopaminergic state characterized by reduced levels of DA receptors in the ventral striatum (Hietala et al. 1994; Volkow et al. 2002; Volkow et al. 2007; Martinez et al. 2005). Recently however, fluctuations from a hypo- to a hyper-dopaminergic state during three weeks of alcohol abstinence in postdependent rats and human alcoholics was also reported suggesting a complex time- and state-dependent regulation of brain DA mechanisms associated with alcohol exposure (Hirth et al., 2016). In this study by Hirth et al. postdependent rats were also characterize by downregulation of D1 receptors but no changes in D2 receptors and enhanced basal DA levels. Some of these features, (i.e., enhanced basal DA levels) were not present in msP rats which implicates also differences between msP and postdependent rats. Most importantly, fMRI studies revealed that addicted abstinent individuals respond to environmental cues predictive of drug availability with a higher degree of activation of the ventral striatum (Braus et al. 2001; Mann et al. 2014; Reinhard et al. 2015). Altogether these findings point to the possibility that msP rats, reproduce neurobehavioral traits common to dependent individuals such as a basal hypodopaminergic state caused by decreased DA receptor expression or function and enhanced sensitivity of the DA system to acute psychostimulant challenge. The exact mechanism regulating the enhanced reactivity of the mesolimbic DA system associated with the post-dependent state has not been elucidated yet. However, alcohol preferring sP rats have been found to show reduced endocannabinoid-mediated inhibition of VTA DA neurons (Melis et al. 2009). This finding has been complemented by recent findings of reduced dialysate levels of N-arachidonoylethanolamine in the CeA of msP rats (Natividad et al. 2017). Considering the anterograde inhibitory role of endocannabinoid signaling, it is likely that these endocannabinoid signaling deficits “disinhibit” VTA DA activation in response to a drug challenge and possibly presentation of stimuli conditioned to drugs of abuse. In addition to dopamine the post-dependent condition is linked to dysfunction of other neurochemical systems including GABA and glutamate (Chefer et al., 2011; Clapp et al., 2008; Herman and Roberto, 2016). This contrast with our microdialysis observation in which no changes in their extracellular levels were detected in the Nac. On the other hand earlier electrophysiological experiments have demonstrated marked alteration in GABA and glutamate transmission in the CeA of msP rats, also resembling a post-dependent state (Herman et al., 2013; Herman et al., 2016). We speculate therefore that in msP changes of GABA and glutamate functions involve some brain areas (i.e., CeA) but not others (i.e., Nac).

In former human addicts, relapse occurs very frequently and is characterized not only by resumption but also rapid escalation of drug use. One may argue that if msP rats represent a post-dependent phenotype for innate reasons, they should rapidly escalate cocaine consumption when drug is made available following abstinence. To test this hypothesis, we trained msP and Wistar rats to self-administer cocaine for 1 or 6 hours per day, using a classical escalation model (Ahmed, Koob 1998)(Koob and Kreek, 2007), to measure whether prolonged daily exposure to the psychostimulant results in enhanced propensity to escalate daily drug consumption. Under these experimental conditions, we observed increased LgA cocaine intake in both rat lines, consistent with previous findings (Koob 1992; de Guglielmo et al. 2013; Matzeu et al. 2016). However, this escalation was more pronounced in msP rats than in the Wistar line. This finding supports the general hypothesis that a post-dependent phenotype -- either resulting from previous exposure to drugs or, as in the case of msP rats, by inherited characteristics -- is associated with enhanced propensity to escalate drug use. One factor that may limit the interpretation of the results is that we used cocaine for the self-administration studies and amphetamine for fMRI and the microdialysis experiments. On the other hand, these two psychostimulants have a lot in common, including their ability to facilitate DA transmission through actions on neuronal terminals in brain regions receiving DAergic innervation (i.e., Nac, Striatum, Prefrontal Cortex etc). This commonality in the mechanisms of action should legitimate, at least to a large extent, our analysis.

Altogether present data point to the possibility that in msP rats genetic factors responsible for excessive alcohol drinking may be also associated with enhanced propensity to develop addictive-like behaviors for psychostimulants. One of this factor could be the single nucleotide polymorphism that msP rats carries at Crh1r locus and that is responsible for the over-expression of CRF1R in these animals. In fact, enhanced CRF1R mediated transmission not only drives excessive alcohol drinking in msP rats but also increases cocaine self-administration and facilitates escalation in heterogeneous rats (Cippitelli et al 2015; Hansson et al 2006; Hansson et al 2007; (Han et al., 2017; Leonard et al., 2017). A remaining open question is whether msP and Wistar rats differ also in their propensity to relapse in response to environmental cues. Based on present results, a reasonable prediction is that msP rats will show higher levels of relapse in response to cues predictive of cocaine availability. This possibility, even though not yet explored, is suggested by findings that msP rats show greater and more persistent reinstatement of alcohol seeking in response to cues predictive of the availability of this drug compared to Wistar rats (Cannella et al. 2016).

In summary, as revealed by MRI data, msP rats show increased functional reactivity to d-amphetamine in the prelimbic cortex, dorsal striatum, and all subregions of the extended amygdala compared to Wistar rats. This effect is associated with increased extracellular DA levels in the NAC shell following amphetamine which may reflect innate heightened reactivity of the mesolimbic DA system to the drug in msP rats. At the behavioral level, these effects appears to correlate with increased exploratory behavior, reduced sensitivity to the reinforcing effects of low psychostimulant doses and increased propensity to escalate cocaine use. These findings resemble clinical observations of reduced dopaminergic receptor-mediated signaling together with augmented reactivity of the DA system to drugs or to drug-related cues in dependent drug users. In previous studies on the effects of alcohol in msP rats we found that this line shows, for innate reasons, characteristics consistent with a post-dependent state. Here, we expand this observation demonstrating that these characteristics generalize to psychostimulants, suggesting that, independently of the substance, common neurobiological and neurochemical events may exists that facilitate the transition from drug use to addiction. Moreover, an intriguing possibility is that heightened fMRI activation of the extended amygdala and striatum in response to a stimulant challenge may predict individual propensity to escalate drug use and to develop addiction.

Acknowledgements

We thank Alfredo Fiorelli for excellent technical assistance. This study was supported by NIH AA014351 (FW and RC) and AA017447 (MR and RC). All authors contributed to the experimental design and data analyses.

Footnotes

Disclosure/Conflict of Interest

The authors report no biomedical financial interests or potential conflicts of interest. A.B., A.G., A.C., A.M., E.D., L.H., N.C., M.U. conducted the experiments and analyzed the data. G.S. analyzed the data. A.B, F.W. and R.C. analyzed the data, supervised the work and wrote the manuscript.

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